Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species.OmpF is one of the major general porins of  Yersinia, a member of the Enterobacteriaceae family, is currently composed of 14 known species: Y. pestis, Y. pseudotuberculosis, Y. enterocolitica Y. aldovae, Y. aleksiciae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. massiliensis, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. similisY. pestis is the etiologic agent of plague while Y. pseudotuberculosis and Y. enterocolitica are known to cause a variety of gastrointestinal symptoms Yersinia are disseminated all over the world in terrestrial and aquatic environments, and associated with many different hosts . Despite recent advances in our understanding of the pathobiology of Yersinia, the molecular-genetic mechanisms by which Yersinia colonizes and adapts to various host or environmental conditions are still poorly understood. In this context, membrane surface molecules are considered the major targets of the membrane-environment interaction.The genus 5 copies per cell) in the outer membrane of the gram-negative bacteria E. coli, differ in their solute selectivity, porin activity and gene expression in response to many environmental factors, such as osmotic pressure, temperature and pH General bacterial porins (GBPs) are one of the most abundant proteins . In total 73 16S rDNA and gyrB sequences were analysed. The sequences were aligned and adjusted to 750 bp for 16S rDNA and to 838 bp for gyrB. Each unique sequence, differing in one or more nucleotide or amino acids sites, was assigned as a distinctive allele, resulting in 31 alleles for 16S rDNA and 48 alleles for gyrB or 21 alleles for GyrB  or GyrB  and to 13 alleles of gyrB (Y. intermedia). The number of allele variants slightly varied from those published previously In this study, the total number of ocations . 16S rDNfor GyrB . The numgyrB concatenated sequences  were clearly grouped into relatively distinct clusters. The intraspecies genetic distance means of these species were up to 0.012. Y. pestis strains clustered tightly with the Y. pseudotuberculosis strains and the distance mean for this group was 0,001. Since, only one Y. similis strain was examined, the genetic distance of that species could not be estimated. Y. similis is a novel species in Yersinia, recently separated from its nearest phylogenetic neighbor Y. pseudotuberculosisY. similis Y239 was clustered with Y. pseudotuberculosis and Y. pestis, forming a distinctive long branch. Strains of Y. enterocolitica were divided into three groups mainly caused by gyrB sequences, while 16S rDNA sequences separated strains into two subspecies , previously described by Neubauer et al., 2000 Y. frederiksenii 2043 did not group with other five isolates of this species. It branched with Y. aleksiciae, Y. bercovieri and Y. mollaretii. Based on these results phylogenetic relations and BLAST (data not shown), Y. frederiksenii 2043 was more closely related to Y. massiliensis. Similar partition was observed for Y. kristensenii, three of which  diverged from the other eight strains  with a genetic distance about 0.055 and clustered with Y. aleksiciae Y159, sharing the genetic distance by 0,005. The data definitely indicated that these uncommon strains of Y. kristensenii and Y. frederiksenii might be members of Y. aleksiciae sp. nov. and Y. massiliensis sp. nov., since, Y. aleksiciae was recently separated from Y. kristenseniiY. massiliensis is more closely related to Y. frederikseniiY. aleksiciae-like and Y. massiliensis-like, respectively. Based on the 16S rDNA-gyrB tree, most Y. intermedia clustered together into one of two branches; four Russian strains  were located on the line leading to the rest Y. intermedia, shared the intraspecies distances up to 0.007.In order to correctly identify each strain examined, a neighbour-joining tree was constructed from the 16S rDNA-equences . Ten YerYersinia strains based on the 16S rDNA-gyrB concatenated tree was in relative agreement with the MLST tree reported previously Y. kristensenii strains  were designated as Y. aleksiciae-like and one Y. frederiksenii strain (2043) was as Y. massiliensis –like. Six Yersinia species  were genetically more homogeneous then the rest of species .Taken together, species identification of ompF gene from all Yersinia strains  the intraspecies genetic distance mean was 0.029, and the groups means of 0.097–0.166. Phylogenetic grouping of Y. enterocolitica ompF genes exactly replicated that of 16S rDNA-gyrB sequences with division in two subspecies, Y. enterocolitica subsp. palearctica  and Y. enterocolitica subsp. enterocolitica (WA220 and ATCC 8081), supported by a high bootstrap value (100%). Interestingly, in both phylogenetic trees, Y. enterocolitica subsp. palearctica clearly splits into two lines , one of them was only formed by Y. enterocolitica strains  isolated in Russian Far-East. Strains of Y. kristensenii formed group X with intragroup distance mean 0.020, and intergroup distance means 0.072–0.183. The Y. bercovieri (group VI), Y. rohdei (group XIV) and Y. aldovae (group IV) were represented by only two strains and the within and between group distance means were up to 0.009 and 0.068–0.188, respectively. The strains of Y. pestis, Y. pseudotuberculosis and Y. similis grouped together (group VIII) with intragroup distance mean of 0.037, and between group distance means being 0.138–0.196. The VIII group splits into two subgroups with bootstrap value of 100%. One of these subgroups included two Y. pseudotuberculosis strains IP32953, IP31758 and Y. similis Y239, while the other-all Y. pestis strains and Y. pseudotuberculosis YPIII. This ompF tree topology did not correlate with the 16S rDNA-gyrB tree branching, possibly indicating interspecies recombination between Y. pseudotuberculosis and Y. similis, or/and diversification of the ompF gene of Y. pseudotuberculosis before emergence of Y. pestis by adaptive evolution.We investigated phylogenetic relationships and recombination of the  strains . The ompmpF gene , which cmpF gene . Though ompF sequences of the remaining species, Y. intermedia, Y. mollaretii, Y. frederiksenii and Y. aleksiciae, exhibited different phylogenetic relationships and produced incongruent molecular phylogenies with the 16S rDNA-gyrB tree. The Y. frederiksenii strains, that were genetically distinct and not closely related to each other according to the 16S rDNA-gyrB tree, split into three groups; two groups (XI and XII) were presented by single strains, and IX group was by strains with intragroup of 0.043 and intergroup from 0.112 to 0.178 distance means. From previously characterized Y. frederiksenii genomic groups Y. frederiksenii IX and XI groups of ompF could corresponded to genomic groups 1b and 1a, respectively, and XII group (Y. massiliensis)-to genomospecies 2. A mixed branching pattern was found in Y. mollaretii and Y. aleksiciae strains. Two Y. aleksiciae strains (Y159 and Y332) grouped together with three Y. mollaretii strains , whereas two others Y. aleksiciae strains (991 and 6266)-with three other Y. mollaretii . Therefore, Y. aleksiciae (groups XVI and XVII) and Y. mollaretii (groups XV and XVIII) strains split into two relatively distinct groups with intragroup distance means up to 0.007, and intergroup distance means of 0.048–0.197. Interestingly, Y. aleksiciae recently isolated from Y kristensenii was more closely related to Y. bercovieri and Y. mollaretii than to Y kristensenii, and that was confirmed by the 16S rDNA-gyrB tree. Previously, based on the concatenated tufA-tufB tree, Y. aleksiciae type strain LMG 22254 was found to be distinct from the Y. kristensenii cluster and clearly grouped with Y. bercovieri and Y. mollaretiiY. intermedia that formed four different groups  with intragroup distance means up to 0.039, and with between groups means being 0.076–0.195. Moreover, XIII group, formed by five Y. intermedia strains  was separated from the rest Y. intermedia groups by a number of genetic clades.The gyrB and ompF phylogenies. A mix branching pattern can be a sign of recombination, whereas in the case of mutation the gene trees look the same ompF gene of all Yersinia groups. We detected four recombination events with brake-points involving three species, Y. intermedia , Y. aleksiciae (groups XVI and XVII) and Y. mollaretii (groups XVIII and XV) . Group I mainly played a parental role in different recombination events, giving ompF variants of Y. intermedia groups II and XIII; other players of the events were not identified in this analysis. It should be noted, that group I strains are most numerous and widely geographically distributed. So it can be supposed, that this ompF variant is more spread and successful in coexistence with mammals including evolutionary newcomers, humans. Acquisition of regions of a successful allele by recombination can be preferred for minor variants (groups II and XIII) when bacterium get into a new niche such as mammals. Interestingly, an extraordinary position of the XIII group on the phylogenetic tree indicates a new origin of the ompF gene not represented by any known Yersinia species. The fact, that this group includes a human isolate (Nr13/84) may be an evidence of occurrence in new niche, human. Noteworthy, it was extremely difficult to reconstruct a scenario of recombination events for all Y. intermedia ompF. This might be a subject of further research, as well as investigation of associations within a specific niche.As mentioned above, some species produced incongruent 16S rDNA- and XV) . From thompF is located in the same region (431–501 bp), corresponding to 6-th β-strand of OmpF. The reconstruction of the ompF tree for the region 1–501 bp produced very similar branch pattern with that of the 16S rDNA-gyrB tree (data not shown). We suspect that significant nucleotide similarity in this region  reflects a strong selective pressure (purifying selection) due to an important functional role of this region as a zone of It is very interesting to note that one of the brake-points of all recombinant Pseudomonas (OprD), Neisseria , Chlamydia (OmpA), and Leptospira (OmpL1) porB2, an allele of porB, arose in meningococci by interspecies recombination between ancestral pathogenic and commensal Neisseria species ompA between a mouse strain of C. trachomatis and a horse strain of C. pneumoniae was documented ompL1 genes, belonging to four different Leptospira species Examples of the homologous recombination in porin genes have been recognized for some bacteria, mainly for naturally transformable species as ompF sequences placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree with the exception of six species, Y. pseudotuberculosis, Y. similis, Y. frederiksenii, Y. intermedia, Y. mollaretii, and Y. aleksiciae. The incongruence of ompF and 16S rDNA-gyrB trees indicated the inter- and intraspecies recombination. Despite extensive recombination events in the Yersinia ompF genes, this seems to happen not so often to remove all phylogenetic signals.The phylogenetic analysis of ompF gene of Yersinia is more divergent than the 16S rDNA and gyrB genes. The nucleotide diversity for all ompF genes  is twofold higher than for housekeeping genes . The common alignment of 73 ompF sequences contain 40% (479/1200 bp) of polymorphic nucleotide sites, which distributed strikingly nonrandom and formed hypervariable and conserved regions . Therefore, there is a strong codon usage bias in the ompF gene in all Yersinia species, as expected for highly expressed genes. This is another reason to assume that the high level of ompF transcription may be also responsible for nonrandom heterogeneity in the gene.To estimate deviation in codon usage, the codon adaptation index (CAI) was calculated for the ompF gene, we estimated the numbers of synonymous substitutions per synonymous site (dS) and nonsynonymous substitutions per nonsynonymous site (dN) and calculated the dS/dN ratio for the ompF gene. If purifying selection has occurred, a gene has a dS/dN>1. Absence of selection should generate dS/dN = 1. A ratio dS/dN<1 indicates diversifying selection or accelerated evolution Yersinia groups with recombination events from analysis and dealt only with six ompF groups of Yersinia . The dS/dN ratio was calculated as an average over all of the codon sites in each ompF group using the Nei-Gojobori method by MEGA 4 of Jukes-Cantor model. Statistical significance was tested by Codon-based Z-test. For all groups we detected approximately identical dS/dN means from 4.224 to 5.748 with p<0.05 of purifying selection. Thus, ompF gene is under strong purifying selection in all six Yersinia groups. Neilsen and Yang method ompF groups. The porin protein structures for these groups have been simulated and sites with weak or strong positive selection have been located on the models  and function-structure constraint . The data can be important for clarification the role of porin's surface exposed loops on bacterial adaptation and development of broad-spectrum Yersinia vaccine antigens and serological methods of diagnostics.Genetic diversity of outer membrane proteins might result from bacterial adaptation to different ecological niches. Porins are surface exposed and their structure strongly reflects the history of multiple interactions with the environmental changes in their ecological niches. The evolution of the Yersinia strains from the collections of Max von Pettenkofer Institute  and Research institute of epidemiology and microbiology, Siberian branch of Russian academy of medical sciences,  were used in this study. Strain selection was intended to include strains of all known Yersinia species with a high degree of diversity. All strains were grown overnight at 30°C or 37°C under aerobic conditions on LB medium. Bacterial DNA was isolated from overnight cultures of the selected strains using Genomic DNA Purification Kit . The DNA concentration was determined by agarose gel electrophoresis. The gels were scanned and the signals were analyzed with the VersaDoc 4000 MP system . Additionally, eleven Yersinia strains for which the genome sequences are available on the GeneBank of NCBI website were analyzed.A total of 65 Yersinia was performed using the primers, BF-20 (5′–ATCACGCGTAAAAATCT-3′) and BR2-22 (5′-CCGCAATATCATTGGTGGT-3′). The expected amplicon size was 1500 bp. The part of gyrB gene was amplified using primers YgyrF (5′-CCCACTTTATACCT-3′) and YgyrR (5′-CCCACTTTATACCT-3′). The expected amplicon size was 980 bp. The ompF gene was amplified using primers Fcds-F (5′-CCCACTTTATACCT-3′) and Fcds-R (5′-CCCACTTTATACCT-3′). These were designed by aligning sequences of ompF genes of Y. enterocolitica 8081 (AM286415), Y. intermedia ATCC 29909 (AALF02000006), Y. mollaretii ATCC 33641 (NZ_AALD02000003) and Y. frederiksenii ATCC 33641 (NZ_AALE02000015). The expected amplicon size was 1100 bp. PCR conditions for all genes were as follows: initial denaturation at 95°C for 5 min followed by 30 cycles each at 94°C for 30 s, 55°C for 30 s, 72°C for s and a final extension step at 72°C for 5 min. PCR products were evaluated on a 1,5% agarose gel stained with ethidium bromide. Unincorporated primers and dNTPs were removed from PCR products with NucleoSpin® Extract II kit (Macherey-Nagel). Purified DNA was sequenced using the dideoxynucleotide chain-termination method with fluorescent ddNTPs from Applied Biosystems on an ABI 310 Prism automated DNA sequencer, in accordance with the manufacturer's instructions. Sequence data for the appropriate loci from Y. bercovieri ATCC 43970 (NZ_AALC00000000), Y. enterocolitica 8081 (NC_008800), Y. frederiksenii ATCC 33641 (NZ_AALE00000000), Y. intermedia ATCC 29909 (NZ_AALF00000000), Y. mollaretii ATCC 43969 (NZ_AALD00000000), Y. pestis 91001 (NC_005810), CO92 (NC_003143), Pestoides F (NC_009381), Y. pseudotuberculosis IP 31758 (NC_009708), IP 32953 (NC_006155), YPIII (NC_010465), Y. rohdei ATCC 43380 (NZ_ACCD00000000), Y. kristensenii ATCC 33638 (NZ_ACCA00000000), Y. ruckeri ATCC 29473 (NZ_ACCC00000000), Y. aldovae ATCC 35236 (NZ_ACCB00000000) were obtained from GenBank (http://ncbi.nlm.nih.gov) and analyzed together with other Yersinia isolates  and non-synonymous  substitution rates in MEGA 4 using the Nei-Gojobori method with Jukes-Cantor correction and SLR ompF sequences were detected with the RDP v3.34 software ftp://molbiol.ox.ac.uk/cu/codonW.tar.Z) software for 11 Yersinia species . As a reference for highly expressed genes, we used the 26 concatenated ribosomal genes for each organism.Nucleotide divergence (Pi) along The novel sequences determined in this study have been deposited in GenBank under accession no. GQ421361-GQ421424; FJ641877-FJ641894; 146 HM142614-HM142721.


Regenerative medicine is the process of replacing or regenerating animal cells, tissues, or organs to restore or establish normal function. This field has the potential to solve the problem of the shortage of organs available for donation and of organ transplant rejection. Recently, application of Complementary and Alternative Medicine (CAM) as a means to accelerate the process of regeneration is a new approach. The CAM therapies offer a natural and cost-effective intervention to change the course of chronic disease and may regenerate failing organ systems.Tremella fuciformis polysaccharide (TF) and successfully used the TF-immobilized conduits to repair a large (15 mm) critical gap defect in rats. Y. Lee et al. found that L-glutamate-induced neurotoxicity could be suppressed by the treatment with constituents of Rhodiola rosea, indicating that the Rhodiola rosea may have therapeutic potential for the treatment of inflammation and neurodegenerative disease. Dr. B.–Y. Yang et al. tried to apply percutaneous electrical stimulation to improve bone remodeling and bone healing in rats. X-ray and micro-CT showed that the electrical treatment could increase the amount of newly formed cranial bone. W. Liu et al. examined the contribution of side population (SP) cells from kidney and bone marrow to the reconstitution of kidney SP pools after ischemia-reperfusion injury (IRI). They found that following renal IRI, kidney SP cells were acutely depleted and then progressively restorated to baseline levels by both self-proliferation and extrarenal source, that is, bone marrow-derived cell homing. Dr. F. Yahya et al. showed that methanol extract of Bauhinia purpurea leaves could exert potential hepatoprotective activity in rats via its antioxidant activity and high phenolic content. T. Jayakumar et al. reviewed the effects of andrographolide, a major bioactive chemical constituent in Andrographis paniculata (Burm.f.) Nees, against cardiovascular disease, platelet activation, infertility, and NF-kB activation. Dr. H.–M. Chiang et al. showed the antioxidant activity of a Neonauclea reticulata water extract against ultraviolet B (UVB) irradiation in human skin fibroblast cell cultures (Hs68) by inhibiting MMP-1, -3, and -9 expressions and increasing levels of collagen activity. Dr. W.–Y. Su et al. developed a biphasic calcium phosphate cement, consisting of α-tricalcium phosphate (α-TCP) and hydroxyapatite (HAP), which is a potential biomaterial for bone repair. C. Y. Ho et al. showed that acupuncture and electroacupuncture could have positive effects on regeneration of median nerve in rats. J. Xiao et al. demonstrated that administration of garlic-derived antioxidant S-allylmercaptocysteine (SAMC) could ameliorate hepatic injury in a nonalcoholic fatty liver disease rat model. Dr. S.–C. Lee et al. investigated the effect of ferulic acid (FA) against peripheral nerve injury. They found that FA appears to promote peripheral nerve regeneration across a 15 mm critical defect gap in the rat sciatic nerve injury model. Suppression of macrophages by FA at the site of peripheral nerve injury may contribute to its nerve growth-promoting capability. The paper by Dr. Y.–J. Chen et al. explores how the Hepatitis B virus-encoded X regulates the expression of epidermal growth factor receptor, an important gene for growth of hepatocytes. Dr. H.–Y. Liu et al. demonstrated the regenerative potentials of deep sea water on osteogenesis, showing that deep sea water could potentially be applied in osteoporosis therapy. Dr. S. –H. Hsu et al. prepared peripheral nerve conduits containing the negatively charged By compiling these articles, we hope to stimulate our readers and researchers to provide continuing efforts to fully understand the effects of CAM therapies on regenerating tissues or organs.Yueh-Sheng ChenYueh-Sheng ChenWei-Chiang LinWei-Chiang LinCheryl MillerCheryl Miller


Accurate identification of isolates belonging to genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations,especially when large blind samples must be analyzed. It is useful to simultaneously identify species in different genera using a single primer pair.We developed a primer pair based on the tuf gene (encoding elongation factor) sequence to identify 56 Gram-positive cocci isolates.The target sequences were amplified from all 56 samples. The sequencing results and the phylogenetic tree derived from the partial tuf gene sequences identified the isolates as three enterococcal species, two lactococcal species, two staphylococcal species, and six streptococcal species, as well as eight isolates that were novel species of the genus Streptococcus. Partial gene sequence analysis of the sodA, dnaK, and 16S RNA genes confirmed the results obtained by tuf gene sequencing.Based on the uniform amplification of the tuf gene from all samples and the ability to identify all isolates at both the genus and species levels, we conclude that the primer pair developed in this research provides a powerful tool for identifying these organisms in clinical laboratories where large blind samples are used. Bacterial species of the genera Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus are Gram positive cocci in the class Bacilli. Enterococcus, streptococcus, and staphylococcus inhabit a wide range of environments, including the gastrointestinal tracts of humans and animals. Lactococcus is a genus of lactic acid bacteria that were formerly included in the genus Streptococcus, and have been widely used in the production of cheese and milk products.Accurate identification of isolates belonging to these genus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations. Because species identification based on phenotypic characterization is time-consuming and can produce ambiguous results -6, molecThe tuf gene, which encodes the elongation factor EF-Tu, is involved in peptide chain formation. It is a ubiquitous and highly evolutionarily conserved part of the core genome, and is more discriminative than the 16S rRNA gene for identifying strains belonging to the genera Enterococcus , StaphylStreptococcus suis (S.suis)serotype 2 strain 9801, S. suis 05ZYH33, one Lactococcus lactis subsp. lactis isolate, 48 cocci isolates that was isolated by our lab between 2006 and 2011, and four cocci strains purchased from the Chinese General Microbiology Culture Collection Center (CGMCC), which included the following: Streptococcus pernyi (S.peryi) CGMCC1.1010, Streptococcus salivarius CGMCC 1.2498, Streptococcus mutans CGMCC 1.2499, and Streptococcus bovis CGMCC1.2502. Streptococcus cremoris (L. lactis subsp. cremoris) CICC 20175 was also purchased from the Chinese Center of Industrial Culture Collection (CICC).The strains examined in this study included Genomic DNA was isolated and purified using a GenElute Bacterial Genomic DNA Kit (Sigma) according to the manufacturer’s instructions. PCR primers complementary to highly conserved regions of the tuf gene were designed . tuf gene nucleotide sequences were submitted under GenBank accession numbers JX436506–JX436520; sodA nucleotide sequences were submitted under GenBank accession numbers JX436496–JX436497; L. lactis subsp lactis strain LVRI001 dnaK sequence was submitted under GenBank accession number 436495; and 16S rRNA nucleotide sequences were submitted under GenBank accession numbers JQ255459–JQ255462.Nucleotide sequences determined in this study were submitted to GenBank  isolates were identified, all of which showed 100% sequence similarity to the tuf gene of S. thermophilus ND03, while a further eight isolates were identified as Staphylococcus warneri (S. warneri), which showed 100% nucleotide sequence similarity to each other, and 99.5% (777/781 bp) similarity to the tuf gene of S. warneri ATCC 27836. Six strains were identified as Staphylococcus hyicus (S. hyicus )and shared 100% identity with each other and 99.9% (705/706 bp) identity with S. hyicus strain CIP 81.58. Seven Enterococcus faecalis  strains were also identified and showed 99.9% identity to the tuf sequence of E. faecalis V583. Five Enterococcus avium (E. avium ) isolates shared 99.6–100% sequence similarity with each other and 98.6% (706/717 bp and 707/717 bp) sequence similarity to E. avium ATCC 14025. Finally, we identified eight isolates as belonging to the genus Streptococcus, but could not make a species identification based on comparison with published tuf sequence data in GenBank or EMBL. These sequences shared 99.0–100% sequence similarity with each other, and the highest tuf gene homology was with S. suis A7 . The levels of partial tuf gene sequence divergence between the isolates and representatives of other Staphylococcus species were in the range 5.1–14.2%.NCBI BLASTN analysis revealed that the 48 isolates collected by our lab included eight Streptococcus uberis strains that have 99.8–100% sequence similarity to each other in the tuf gene. The nucleotide sequences of these tuf genes shared approximately 99.8% nucleotide similarity with that of The tuf gene diversity was calculated using Kimura’s two-parameter mode using Mega 5.0. At the genus level, the tuf sequence diversity among different species within the genus Streptococcus ranged from 1.1–12.9%. Sequence diversity ranged from 9–12.3% among different species in the genus Enterococcus. For genus Staphylococcus, diversity ranged from 4.7–10.1%.Because the isolates might be misidentified when using a single gene sequence for identification, we used a second partial gene sequence to confirm the result obtained by tuf gene analysis. The sodA gene was used for confirmation of the isolates identified as belonging to the genera Staphylococcus and Enterococcus. For the isolates in the genus Streptococcus, a partial dnaK sequence was amplified and sequenced. The sequencing results confirmed the results obtained from the partial tuf gene analysis.S. warneri strains used in this research showed 99.5% (777/781 bp) nucleotide sequence (nt) similarity to S. warneri ATCC 27836, it also showed 99.5% (767/771) nt sequence similarity to Staphylococcus pasteuri (S. pasteuri )FI64. For this reason, the partial sodA sequences were then used for analysis. The sodA gene of the S. warneri strains isolated in our lab were found to show 99.8% (428/429 bp) sequence similarity to the sodA gene of S. warneri CIT S00-147. However, they were clearly distinguished from S. pasteuri strain CIP 103540 T, with a nucleotide sequence similarity of only 94.3% (396/420 bp).When S. pernyi CGMCC1.1010 was obtained from the CGMCC. In a recent work by Cui-Fang et al., this strain was suggested to be an enterococcal species [S. pernyi CGMCC1.1010 has 98.9% nucleotide identity (792/801 bp) to that of E. mundtii strain ATCC43186. Furthermore, analysis of the partial sodA sequence revealed that sodA of S. pernyi CGMCC1.1010 has 99.8% nucleotide identity (437/438 bp) to that of E. mundtii strain ATCC 43186. Thus, we confirmed that CGMCC1.1010 is actually an E. mundtii strain.The strain  species . Based oStreptococcus minor (S. minor )strain LMG 21735  and S. minor strain ON59 . Because isolates that show ≤ 98.7% 16S rRNA sequence similarity are always considered members of a different species [Streptococcus parasuis. Further biochemical research is being conducted to classify and confirm this suspected novel species.Phylogenetic analysis was carried out using the neighbor-joining method .Reliable discrimination between closely related species depends on the variability of the target gene. A high level of variability might be helpful for good discrimination between species, but it can also be a disadvantage because of instability of species-specific signatures and difficulties in developing reliable primers or probes. Because of the high level of variability, the higher the discriminating power, the higher the proportion of strains that are not amplified using a single primer pair -34. In tThe tuf gene-based primer pair designed in the present study may be helpful for the accurate detection and identification of Enterococcus, Staphylococcus, Lactococcus, and Streptococcus species, as well as related genera of medical interest.In conclusion, this study confirms that tuf is a good alternative molecular marker for both phylogenetic analysis and species identification of clinical isolates when large blind samples are used. It should be applied to phylogeny as a first-line genomic technique.S.suis: Streptococcus suis; S.peryi: Streptococcus peryi; S. thermophilus: Streptococcus thermophilus; S. warneri: Staphylococcus warneri; S. hyicus: Staphylococcus hyicus; E. faecalis: Enterococcus faecalis; E. avium: Enterococcus avium; S. pasteuri: Staphylococcus pasteuri; E. mundtii: Enterococcus mundtii; S. minor: Streptococcus minor.Tuf: Elongation factor Tu; Authors have no competing interests.Xuerui Li, Juanxing and Baoyu Li co-worked on data collection and organisation, performed statistical analysis of the data and contributed to writing and interpretation of the manuscript. Pu wang wrote the manuscript. Jixing Liu contributed to the design and writing of the manuscript. All authors have read and approved the manuscript.Sources and gene accession numbers of the bacterial reference strains used in this study.Click here for file


Enterobacter cloacae species includes an extremely diverse group of bacteria that are associated with plants, soil and humans. Publication of the complete genome sequence of the plant growth-promoting endophytic E. cloacae subsp. cloacae ENHKU01 provided an opportunity to perform the first comparative genome analysis between strains of this dynamic species. Examination of the pan-genome of E. cloacae showed that the conserved core genome retains the general physiological and survival genes of the species, while genomic factors in plasmids and variable regions determine the virulence of the human pathogenic E. cloacae strain; additionally, the diversity of fimbriae contributes to variation in colonization and host determination of different E. cloacae strains. Comparative genome analysis further illustrated that E. cloacae strains possess multiple mechanisms for antagonistic action against other microorganisms, which involve the production of siderophores and various antimicrobial compounds, such as bacteriocins, chitinases and antibiotic resistance proteins. The presence of Type VI secretion systems is expected to provide further fitness advantages for E. cloacae in microbial competition, thus allowing it to survive in different environments. Competition assays were performed to support our observations in genomic analysis, where E. cloacae subsp. cloacae ENHKU01 demonstrated antagonistic activities against a wide range of plant pathogenic fungal and bacterial species.The  Enterobacter cloacae is a gram-negative Proteobacterium belonging to the Enterobacteriaceae family. Within this family, Enterobacter is most closely related to, and is grouped in a sub-clade with, Klebsiella. The two groups of bacteria diverged from a sub-clade consisting of Escherichia, Citrobacter and Salmonella [monella .E. cloacae species comprises an extremely diverse group of bacteria that has been found in diverse environments, ranging from plants to soil to humans. Plant pathogenic strains of E. cloacae have been reported to cause Enterobacter bulb decay in onion plants and bacterial wilt in mulberry [E. cloacae strains have been shown to colonize and benefit plant growth in various crops, such as soybean, cucumber, corn, rice and ginger [E. cloacae has antagonistic effects against the oomycete pathogen Pythium ultimum [Fusariummoniliforme and Fusarium oxysporum [Ralstonia solanacearum [E. cloacae are considered to be plant growth-promoting rhizobacteria (PGPR). The genomic features that underlie the antagonistic characteristics have been demonstrated in previous genome analyses of model PGPR species, such as the gram-negative Pseudomonas and the gram-positive Bacillus [Enterobacter spp.The mulberry ,3; endopd ginger ,5. Previ ultimum , the funxysporum  and the nacearum . Additioacillus ,10,11, bE. cloacae is best known as a human opportunistic pathogen that is commonly found in hospitals and causes a wide range of infections, such as lower respiratory tract infections, urinary tract infections and meningitis [E. cloacae is clinically significant, particularly because its strains usually carry multiple antibiotic resistance genes [E. cloacae subsp. cloacae ATCC13047, a classic strain isolated in the last century from human brain fluids, but no detailed analysis or supporting data were reported [ningitis . Outbreace genes ,14. The reported .Enterobacteriaceae such as Escherichia, Salmonella and Yesinia, which are well-known for their association with pathogenicity in humans and animals, there is scarcity of genomic data for Enterobacter. Eight complete Enterobacter genomes have been reported to NCBI (http://www.ncbi.nlm.nih.gov/genome/1219): E. aerogenes KCTC2190, E. cloacae subsp. cloacae ATCC13047, E. cloacae subsp. dissolvens SDM, E. cloacae subsp. cloacae NCTC9394, Elignolyticus.  SCF1 (named as E. cloacae SCF1 in NCBI database), E. asburiae LF7a, E. cloacae EcWSU1 and Enterobacter sp. 638. These genome sequencing projects mostly emphasized the potential for the use of Enterobacter in industrial applications, such as lignin degradation [Enterobacter sp. 638 [Enterobacter genomes belong to E. cloacae, which include human opportunistic pathogens E. cloacae subsp. cloacae NCTC9394 and E. cloacae subsp. cloacae ATCC13047 [E. cloacae EcWSU1 [E. cloacae subsp. dissolvens SDM [Compared with other genera in the family of radation  and 2,3-radation ,18,19. ATCC13047 , a plante EcWSU1  and a 2,E. cloacae subsp. cloacae ENHKU01 and the available complete genome sequence data from different strains of E. cloacae [E. cloacae strains that originated from diverse environments. In this study, we identified crucial conserved genomic factors that support the antagonistic functions of E. cloacae. Genomic factors differentiating the various strains of E. cloacae were also investigated. Also, this study is the first comprehensive comparative genome analysis for E. cloacae genomes.Using the complete genome sequence of the plant growth-promoting endophytic  cloacae ,19,20,21E. cloacae subsp. cloacae ENHKU01 was isolated from a pepper plant infected by R. solanacearum in Hong Kong in May 2010. Three single colony subcultures were performed to obtain a pure isolate on TTC  medium w/v 1.5% agar plates at 28°C. An inoculation and re-isolation experiment using 4-week old tomato and pepper seedlings was carried out to confirm its endophytic characteristic in plants. Unlike R. solanacearum, the bacterial isolate did not cause wilting symptoms in these plants following inoculation. Inoculation on potato tubers and onion bulbs also indicated that the isolate did not cause disease in these plants. Sequencing and BLAST analysis of partial 16S rRNA and housekeeping genes  [E. cloacae and was most closely related to E. cloacae subsp. cloacae ATCC13047.0, rpoB)  was condE. cloacae subsp. cloacae ENHKU01 was performed as described in Liu et al., 2012 [de novo shotgun sequencing and paired-end sequencing strategies were applied to produce the whole genome sequence of ENHKU01 using the 454 GS Junior platform . Newbler Assembler software  was used to construct a draft genome with one scaffold containing 36 contigs [E. cloacae subsp. cloacae ENHKU01, sequence gaps were filled by PCR, primer walking and Sanger sequencing.Whole genome sequencing and annotation of l., 2012 . In brie contigs . To compftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Enterobacter_cloacae_ENHKU01_uid172463/.Gene annotation and analysis was performed using NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) . AnnotatEnterobacter species: E. aerogenes KCTC2190 (GenBank accession CP002824), E. cloacae subsp. cloacae ATCC13047 , E. cloacae subsp. dissolvens SDM (GenBank accession CP003678), Elignolyticus.  SCF1 (named as E. cloacae SCF1 in NCBI database) (GenBank accession CP002272), E. asburiae LF7a , E. cloacae EcWSU1 (GenBank accession CP002886 and CP002887) and Enterobacter sp. 638 (GenBank accession CP000653 and CP000654) were obtained from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/). E. cloacae subsp. cloacae NCTC9394 sequence data were not available for download; thus, it was not included in this study. Scaffolds of the Enterobacter species involved in this study were also uploaded to the Rapid Annotations using Subsystems Technology (RAST) server for SEED-based automated annotation, whole-genome sequence-based comparative analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway for comparative analysis [Efficient Database framework for comparative Genome Analyses using BLAST score Ratios (EDGAR) was used for core genome, pan genome and singleton analysis, and Venn diagram construction using E. cloacae subsp. cloacae ENHKU01 as a reference genome [For comparative genome analysis, complete genome sequence data of Enterobacter spp. Scaffolds and genome data of the chromosome of eight Enterobacter genomes and three Pantoea genomes: Pantoea sp. At-9b (NC_014837), Pvagans.  C9-1 (NC_014562) and Pananatis.  LMG20103 (NC_013956), were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). Gene clustering was performed using OrthoMCL 2.0 under default parameters [Enterobacter spp. by MrBayes v3.2 using the WAG model. Ten million generations were performed with four chains; burn-in was set to 10,000 generations [An in-house pipeline has been developed for the phylogenomic analysis of rameters . A minimrameters . The corerations .E. cloacae subsp. cloacae ENHKU01 was used as a bait to search for orthologs in the NCBI database by BLASTP/ BLASTX using an E-value cut-off of 1e-10. Protein sequences of the top 100 hits were obtained. MEGA5 was used to perform phylogenetic analysis, multiple alignments of protein sequences were built by CLUSTALW and a 100 bootstrap replicate Neighbor-joining tree was constructed for phylogenetic analysis [Phylogenetic analysis. To analyze the phylogeny of specific genes-of-interest, partial- or full-length CDS of analysis .http://img.jgi.doe.gov) with 1350 finished bacterial genomes. T6SS clusters containing core component genes COG0542 (clpV) with at least four of the following loci: COG3516 (ImpB), COG3517 (ImpC), COG3519 (VasA), COG3520 (VasB) and COG3522 (ImpJ/VasE) were included for further analysis. T6SS clusters with fewer than five core component genes were discarded. As a result, a total of 346 T6SS clusters were identified in 230 bacterial genomes, mostly belonging to the five subdivisions of Proteobacteria. Two T6SSs of ENHKU01 were manually identified and reconfirmed by BLAST and included in the analysis. MEGA5 was used to perform phylogenetic analysis for the ClpV orthologs in a total of 348 T6SS [Phylogenetic analysis of the Type VI Secretion System (T6SS) was performed using a similar strategy as described in previous studies with modifications . T6SS co348 T6SS . MultiplE. cloacae subsp. cloacae ENHKU01 against various plant bacterial and fungal pathogens. All biological samples involved in this study were collected within a 200 km zone in Hong Kong SAR and Guangdong, China. Plant pathogenic fungi Alternaria sp., Choanephorainfundibulifera, Colletotrichumcapsici, DidymellabryoniaeFusarium oxysporum, , Sclerotinia sclerotiorum and Sclerotinia rolfsii were isolated from infected host plants and purified by subsequent subculture on potato dextrose agar (PDA) plates at 28°C; next, pure isolates were obtained and strain identification was confirmed by cloning and sequencing . For fungal antagonistic assays, each challenging fungus was grown for 4 days at 28°C. A sample composed of 0.5 cm of agar with hyphae was cut and placed at 2 cm from the edge of a new potato dextrose agar (PDA) plate. An overnight culture of ENHKU01 was streaked across the middle of the plate [E. cloacae subsp. cloacae ENHKU01. Fungal growth was monitored for two weeks. Three independent experiments were performed for each E. cloacae subsp. cloacae ENHKU01-fungal antagonistic assay.Bioassays were designed to investigate the antagonistic activities of he plate . ControlRalstonia solanacearum, was isolated from the same pepper plant as the E. cloacae subsp. cloacae ENHKU01 isolate. Three single-colony subcultures were performed to obtain a pure isolate on TTC medium w/v 1.5% agar plates at 28°C. Identification of R. solanacearum was confirmed by cloning and sequencing of partial 16S rRNA and fulfilling Koch’s Postulates  [E. cloacae subsp. cloacae ENHKU01- R. solanacearum competition assays were performed in both planktonic and biofilm cultures. To prepare for the competition assay, bacterial cultures were grown overnight in TTC medium and adjusted to OD600=0.1(~1 x 108 c.f.u/ml). For the planktonic culture, equal volumes (25 ml) of ENHKU01 and R. solanacearum were mixed in a 1:1 ratio in a 250 ml flask and incubated in a shaking incubator (250rpm at 28°C). One milliliter of culture was collected at 2, 4, 6 and 24 hours after incubation for quantification. For the biofilm culture, ENHKU01 and R. solanacearum were mixed in a 1:10 ratio. 20 µl of ENHKU01 culture was placed on a TTC w/v 1.5% agar plate and incubated at 28°C. Biofilm samples were collected for quantification using 1 ml pipette tips and re-suspended in water after 24 hours of incubation. Controls consisted of samples containing only ENHKU01 or R. solanacearum for both planktonic and biofilm competition assays. For quantification , serial dilutions were prepared for each sample to a desirable concentration and plated on TTC w/v 1.5% agar. C.F.U.s were counted after incubation at 28°C for 48 hours. ENHKU01 and R. solanacearum can be distinguished by morphological differences in size, color and texture. Four replications were performed for each assay.The bacterial isolate, ng Kong) . E. cloaEnterobacter spp. using 16S rRNA and house-keeping genes has often been inconsistent [E. cloacae in previous studies [Enterobacterspp. and three Pantoeaspp. The four functionally distinctive strains of E. cloacae (the plant growth promoting E. cloacae subsp. cloacae ENHKU01 (ENHKU01) ,the plant pathogen E. cloacae EcWSU1 (EcWSU1), the opportunistic human pathogen E. cloacae subsp. cloacae ATCC13047 (ATCC13047) and the 2,3-butanediol producing E. cloacae subsp. dissolvens SDM (SDM)) were clustered together, and this observation is consistent with previous studies of the phylogenetic relationships of E. cloacae [The classification of nsistent . It has  studies . Phyloge cloacae  were predicted, with 87% being connected to Clusters of Orthologous Groups (COGs). The genome size, total number of genes and predicted CDS of ENHKU01 is slightly smaller but similar to that of EcWSU1 and SDM, while ATCC13047 appears to have a larger genome of ~5.31 Mb. The genome expansion of ATCC13047 is due to the addition of two plasmids and more than 20 genomic variable regions in its chromosome. EcWSU01 contains one plasmid, and SDM lacks plasmids. There is an absence of sequence similarity between the plasmids in different strains of E. cloacae. The genomic GC content of the strains ranges between 54.5-55.1%. MAUVE analysis showed an overall collinear relationship across E. cloacae strains, despite a large scale chromosomal reorganization that occurred in ATCC13047 that was conferred by a single recombination event and resulted in the inversion of a genomic region , which are all species commonly known as human pathogens or opportunistic pathogens. ATCC13047 is believed to acquire multiple heavy metal resistance genes horizontally from other human microflora associated bacteria, thereby contributing to the adaptation and fitness of the bacteria in heavy metal-rich environments, such as sewage [The virulence genes of pathogenic bacteria are often associated with pathogenicity islands that encode a Type III secretion system (T3SS) or a Type IV secretion system (T4SS) acquired by horizontal gene transfer ,42. Thesectively . T4SS iss sewage .E. cloacae EcWSU1 was reported to be a plant pathogen that causes Enterobacter bulb decay in the onion, no T3SS or T4SS was observed in either the plasmid or the chromosome. It remains largely unclear how EcWSU1 became a plant pathogen.The pathogenesis of many plant pathogens often involves T3SS and its associated effector proteins . AlthougProteobacteria and are known to be critical in adhesion and the specific binding to tissues of preferred hosts [E. cloacae and observed significant diversity. Nine to thirteen fimbrial protein-encoding loci were identified in each strain, but only four of them were conserved across all four E. cloacae strains  [Enterobacter species [Enterobacter CFA/I fimbriae is most closely related to Escherichia coli  fimbrial proteins (ECL_00070-00074), has drawn our attention. The locus is located in a cluster of genes encoding the virulence related resistance-nodulation-division (RND) efflux system within a variable region that is absent in the plant-associated ENHKU01 and EcWSU1strains . CFA/I fi (ETEC) . BLAST a species  , an essential growth element from soil. These microbes produce higher affinity siderophores and indirectly suppress the growth of competing fungi, which produce lower affinity fungal siderophores, and non-siderophore producing bacteria [Enterobacter spp. In addition, E. cloacae strains possess an extra siderophore assembly kit for aerobactin siderophore biosynthesis, which is absent in certain other Enterobacter spp. , as they have antagonistic effects against other microorganisms and are used commercially as biological control agents for various fungal pathogens [The ability of microbes to produce a wide range of antimicrobial compounds, including lytic agents, antibiotics, bacteriocins, protein exotoxins and other secondary metabolites, is critical to their success in antagonistic activities. In bacterial-fungal interactions, cell wall hydrolases, such as certain proteases and chitinases, are produced and secreted extracellularly by bacteria to target fungal cell walls ,59,60. Cn system , while trcescens ,63  [The T6SS is widely distributed in gram-negative bacteria. A T6SS usually involves approximately 15 conserved genes in a cluster with genes encoding a functional secretory apparatus that penetrates cell membranes and translocates effector proteins into their eukaryotic hosts or recipient cells ,73. Earlic cells . Within duction) ,82,83 anduction) ,85  ,83.Enterobacter, but the T6SS clusters within a genome are not associated with one another   . In clad Ep1/96 ; also, tClade II . Our obsE. cloacae strains is expected to allow the bacterial strain to enhance the survival of non-pathogenic strains in their habitats, thus competing with other microbes for the colonization of hosts [E. cloacae strains possessing more than one distinct T6SS cluster, such as ATCC13047 and ENHKU01, are likely to have fitness advantages in a borader range of habitats and be able to survive in different environments.The presence of T6SS in of hosts . E. cloaE. cloacae has been revealed in this study, and we further hypothesized that E. cloacae has advantages in microbial competition against other microbes within its environmental niche. To investigate the effect of antagonistic activities in E. cloacae strains further, we performed bacterial-fungal and bacterial-bacterial antagonistic assays using the plant growth-promoting endophytic ENHKU01 as a model. As expected, ENHKU01 showed significant antagonistic effects against a wide range of fungal species: Collectotricumcapsici. Sclerotinia sclerotiorum, Alternaria sp., Didymellabryoniae, and Fusarium oxysporum by suppressing their growth at different levels Click here for additional data file.Data S2A Venn diagram of the data generated by EDGAR .(XLSX)Click here for additional data file.Data S3E. cloacae strains using E. cloacae subsp. cloacae ENHKU01 as the reference genome [List of singletons of ENHKU01, EcWSU1, ATCC13047 and SDM generated by comparing the genomes of the four of e genome .(XLSX)Click here for additional data file.Data S4Enterobacter, corresponding to List of genes associated with Type VI Secretion Systems in (XLSX)Click here for additional data file.Figure S1The comparative analysis of the CFA/I Fimbrial cluster.(DOCX)Click here for additional data file.Table S1Enterobacter by EDGAR.In brief, E. cloacae subsp. cloacae ENHKU01 was selected as reference genome and its gene contents were taken as the base set for stepwise comparison of the seven Enterobacter genomes involved in this study. For each round of all-against-all comparison based on a bidirectional best BLAST hit, every gene in a Enterobacter genome that has no ortholog in the base set was added to the reference set and compared with the next genome. The process was repeated step by step to obtain the pan-genome of Enterobacter [Pan-genome analysis of obacter .(XLSX)Click here for additional data file.Table S2Enterobacter.List of genes involved in potential antibiotic resistance, multidrug resistance and multidrug efflux systems in (XLSX)Click here for additional data file.Table S3E. cloacae subsp. cloacae ENHKU01 against plant pathogenic fungal species.Table shows antagonistic activities of (DOCX)Click here for additional data file.


To the Editor: Spread of multidrug-resistant Neisseria gonorrhoeae is a major public health concern. Effective antimicrobial therapy is a key element in gonorrhea control. However, N. gonorrhoeae has developed resistance to multiple classes of antimicrobial drugs, including β-lactams, tetracyclines, and fluoroquinolones . The woman visited a clinic in Kyoto for a routine examination for sexually transmitted infections in January 2009. Although she had no obvious symptoms or signs, a throat sample collected on her first visit yielded a positive result for N. gonorrhoeae by the strand displacement amplification test , but a vaginal sample taken at the same time was negative. After 2 weeks, another throat sample was positive for N. gonorrhoeae when cultured on Thayer-Martin medium, and the patient subsequently received 1 g ceftriaxone intravenously. Her pharyngeal sample was also N. gonorrhoeae positive by strand displacement amplification test on the third visit 2 weeks later, and further ceftriaxone treatment was prescribed. However, a culture for test of cure was not conducted because reinfection was considered. A negative result was finally obtained in April 2009.We identified a novel ceftriaxone-resistant N. gonorrhoeae. Susceptibility was determined by the agar dilution method  classified the bacterium as N. gonorrhoeae H041, multilocus sequence typing characterized the strain as ST7363 (N. gonorrhoea multiantigen sequence typing (NG-MAST) was also performed  among multilocus sequence typing-ST7363 N. gonorrhoeae strains (n = 81) .To characterize the ceftriaxone-resistant N. gonorrhoeae, H041, is closely related to the ST7363 cefixime-resistant N. gonorrhoeae. Therefore, we compared SpeI-digested genomic DNA banding patterns of strain H041 with those of other N. gonorrhoeae strains by using pulsed-field gel electrophoresis as described  (SpeI digested H041 genomic DNA was similar to that of other ST7363 strains and indistinguishable from that of cefixime-resistant but ceftriaxone-susceptible NG0207 (Molecular typing suggested that the novel ceftriaxone-resistant e NG0207 .N. gonorrhoeae, isolated from a pharyngeal specimen from a female commercial sex worker. At 2 µg/mL, the MIC was 4-fold higher than that of the previously reported ceftriaxone nonsusceptible strain (N. gonorrhoeae colonization of the pharynx may just be tempory because the pharynx is not an ideal site for N. gonorrhoeae growth. From the routine examinations of commercial sex workers during January–March 2009, 40 N. gonorrhoeae were isolated in the clinic, but no other ceftriaxone-resistant strains were isolated. There is no evidence of dissemination of this strain in Kyoto.We describe the emergence of ceftriaxone-resistant N. gonorrhoeae, and it might originate from an ST7363 cefixime-resistant N. gonorrhoeae clone. There are several possible mechanisms for the acquisition of resistance, including formation of a new mosaic type penA allele as penA-X cefixime resistance and acquisition of an extended-spectrum β-lactamase gene. The H041 strain did not produce β-lactamase in a nitrocephin test. Further molecular analysis is needed to elucidate the precise mechanism of the ceftriaxone resistance of the H041 strain.Three independent molecular subtyping methods indicated that the ceftriaxone-resistant H041 strain was N. gonorrhoeae raises concerns for controlling gonorrhea because ceftriaxone is widely recommended and the first-line treatment for gonorrhea around the world. N. gonorrhoeae has a potential to gain an extraordinarily high MIC to ceftriaxone. Surveillance for ceftriaxone-resistant N. gonorrhoeae should be strengthened.The emergence of ceftriaxone-resistant


Veillonella spp. rarely cause infections in humans. We report a case of Veillonella endocarditis documented by isolating a slow-growing, gram-negative microbe in blood cultures. This microbe was identified as the newly recognized species Veillonella montpellierensis (100% homology) by 16S RNA gene sequence analysis. Veillonella are anaerobic, gram-negative cocci, part of the normal flora of the mouth, gastrointestinal tract, and vaginal tract. Veillonella dispar, V. atypica, and V. parvula have been cultured from human specimens. They are infrequently isolated in human infections. Rarely, Veillonella species have been the only etiologic agents identified in serious infections such as meningitis, osteomyelitis, prosthetic joint infection, pleuropulmonary infection, endocarditis, and bacteremia. A new species, V. montpellierensis, has recently been isolated from the gastric fluid of a newborn and from the amniotic fluid of 2 women  scan. Urine culture yielded 2 and anaerobic atmosphere and resulted in small colonies. This slow-growing microbe was lost after 2 subcultures, and no isolate is available for further description. The isolate retrieved in the blood culture was identified by 16S rRNA gene sequence analysis. The template DNA was prepared from a few colonies that were isolated on the blood agar incubated anaerobically. DNA was extracted by using Fastprep DNA extraction kit  according to the manufacturer's recommendations and was subjected to polymerase chain reaction (PCR) targeting the 16S rRNA gene as previously described (V. montpellierensis (GenBank accession no. AY244769) and was already reported (GenBank accession no. AY244769) in a previous article (Veillonella strain (that was isolated from our patient) was first identified as "candidatus V. atypica" since the sequencing of the amplicon disclosed 94% sequence similarity with that of V. atypica  yielded a slow-growing, gram-negative microbe. Blood was subcultured onto Columbia agar with 5% sheep blood  under 5% COPropionibacterium acnes, and Bacteroides fragilis group (Veillonella species was the sole pathogen and the first due to V. montpellierensis. Characteristics of the 7 Veillonella endocarditis patients are summarized in the Veillonella endocarditis were due to either V. dispar (V. parvula (V. alcalescens (V. parvula (Veillonella spp. had also been isolated from intravenous drug users with polymicrobial endocarditis (V. parvula was isolated from a lung abscess in a patient with echocardiographic vegetations, but blood cultures were negative (Veillonella species, penicillin has been the antimicrobial agent of choice (According to the modified Duke criteria , our patis group . We desc. dispar ,10, V. p parvula , or V. aalescens –8, curre parvula . One pat parvula ,11. Veilcarditis ; V. parvnegative . We coulf choice . These pf choice . We treaVeillonella species named V. montpellierensis by Jumas-Bilak et al. (V. montpellierensis is pathogenic for humans and may be included as a new agent of endocarditis caused by fastidious pathogens.Our isolate has recently been compared with 3 other isolates and classified as a new k et al. . We demoVeillonella spp. identified by PCR amplification and sequencing of 16S rDNA gene and the first case of endocarditis due to V. montpellierensis. This case reemphasizes the usefulness of molecular methods in identifying fastidious microorganisms and in describing new clinical entities (We report here the seventh case of endocarditis due to entities .


Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films.Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/μL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections.Ten-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of . falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05).Reported findings are based on 1,878 PPlasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections.These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between  Plasmodium species and estimation of parasite densities. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics such as size, shape, pigmentation, besides the size and shape of the parasitized red blood cells and the inclusions therein [Plasmodium species already exist to aid microscopic diagnosis of malaria infections [Malaria diagnosis in most settings is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. This enables parasite detection, identification of  therein ,2. Medicfections .Plasmodium species traditionally associated with human infections are Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. However, Plasmodium knowlesi associated with long tailed macaque monkeys has of late crossed over to humans and is now considered by some to be the fifth human malaria [Plasmodium species differ with respect to their biology, clinical symptoms, and treatment requirements [Plasmodium infections is of critical importance with regard to formulation and deployment of effective intervention strategies in endemic regions [ malaria ,4. Sinceirements ,6; accurPlasmodium falciparum is the most prevalent malaria infection in Africa and accounts for the largest proportion of clinical cases [Plasmodium malariae infections are frequently found in sympatry with P. falciparum infections [P. ovale is thought to be highest in sub-Saharan Africa. However, its prevalence is deemed to be lower compared to P. malariae[P. vivax in sub-Saharan Africa [al cases ,8. Plasmfections ,9-11, bufections . The bur malariae. The nean Africa ,14 excepn Africa .Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa stained blood films.This work explores misclassification of four Plasmodium species, and parasite counting techniques. The practice-based component focused on examination of reference malaria slide sets under the guidance of expert microscopists.Ten-day remedial malaria microscopy courses were conducted by Kenya Medical Research Institute/US Army Medical Research Unit-Kenya (KEMRI/USAMRU-K), Malaria Diagnostics Centre (MDC) and Kintampo Health Research Center (KHRC), Ghana from 2008 to 2010 as part of a multilateral capacity building strategy for laboratory personnel in public health and research institutions. Training sessions had both theory-based and practice-based components on parasite detection (presence or absence), morphological characterization of P. falciparum, P. malariae, P. ovale and P. vivax infections with parasite densities ranging from 1,000 – 30,000 parasites/μL. Each slide had a thick and thin film stained using buffered 3% Giemsa solution (Sigma-Aldrich GS1L-1 L) for 60 minutes and integrity of the films maintained with microscope cover-glasses (Fisher Scientific 12-543-D) and DPX mountant (Polysciences 13512). Parasitological validation was conducted via multiple independent examinations of coded sets by expert microscopists in addition to Plasmodium genus and species-specific PCR assays.Slide sets used for training and proficiency assessments consisted of P. falciparum and non-falciparum infections. Pre and post-training test sets were coded differently and participants allowed five minutes to examine each slide and report their findings. Results were evaluated, feedback provided, and hands-on revision exercises conducted under the guidance of expert microscopists.Assessments in species identification were by way of pre and post-training examination of 20 slides sets with the above mentioned species distributed equally between P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results were collated and outcomes per species categorized as false negative (FN), positive without speciation (PS), P. falciparum, P. malariae P. ovale, P. vivax and mixed infections (MX). Findings per category were converted to proportions and differences within and between Plasmodium infections analysed using Marascuilo Chi square test for multiple proportions with post hoc comparisons. Significant differences within species were X2 values ≥ 12.59, df 6, p < 0.05 and between species X2 values ≥ 7.82, df 3, p < 0.05 [1,878 p < 0.05 .P. falciparum infections as PS was significantly higher compared to misclassification as FN, misclassification as P. ovale, misclassification as P. vivax in addition to misclassification as MX (p < 0.0001). Misclassification as P. malariae was significantly higher compared to misclassification as FN together with misclassification as P. ovale (p < 0.005) while misclassification as MX was significantly higher compared to misclassification as P. ovale (p = 0.029). There were significant reductions in post-training misclassifications except for residual misclassification as P. ovale which was marginally lower and residual misclassification as MX which became marginally higher compared to pre-training results (p > 0.05). Residual misclassification as PS was significantly lower compared to residual misclassification as P. ovale, residual misclassification as P. vivax besides residual misclassification as MX (p < 0.005). Reductions post-training misclassifications significantly improved accurate identification of P. falciparum infections compared to pre-training results , misclassification as MX significantly lower compared to the other remaining misclassifications (p < 0.0001) while misclassification as PS was significantly higher compared to misclassification as P. vivax (p = 0.012). There were significant reductions in post-training misclassifications except for residual misclassification as P. ovale which was marginally lower along with residual misclassification as MX which became significantly higher compared to pre-training results (p < 0.0001). Residual misclassification as PS was significantly lower compared to other residual misclassifications (p < 0.05) except residual misclassification as FN. Reductions in post-training misclassifications resulted in a significant improvement in accurate identification of P. malariae infections , misclassification as FN significantly higher compared to misclassification as P. falciparum together with misclassification as P. vivax (p < 0.005) whereas misclassification as PS was higher than misclassification as P. falciparum (p = 0.004). There were significant reductions in post-training misclassifications except for residual misclassification as P. vivax which became marginally higher and residual misclassification as MX which became significantly higher compared to pre-training results. Notably, residual misclassification of P. ovale infections as P. vivax became the most common misclassification compared to other residual misclassifications (p < 0.05). This was followed by misclassification as P. malariae which became significantly higher than residual misclassification as FN besides to residual misclassification as PS (p < 0.05). The observed reductions in some of the post-training misclassifications contributed to a significant improvement in accurate identification of P. ovale infections  whereas misclassification as FN was significantly higher compared to misclassification as P. falciparum together with misclassification as P. malariae (p < 0.05). There were significant reductions in post-training misclassifications except for residual misclassifications as P. ovale along with residual misclassification as MX which became significantly higher compared to pre-training results. Post-training misclassification as P. ovale was the most common residual error (p < 0.0001) followed with residual MX when compared to the rest of the residual misclassifications (p < 0.005) except residual misclassification as P. malariae. The reductions in post-training misclassifications resulted in a significant improvement in accurate identification of P. vivax infections . Misclassification of P. falciparum infections as P. malariae was equally lower compared to similar misclassification on P. ovale and P. vivax infections (p < 0.05) and misclassification of P. falciparum infections as P. ovale significantly lower compared to same misclassification on P. malariae and P. vivax infections (p < 0.001). Misclassification of P. falciparum infections as P. vivax was significantly lower compared to equivalent misclassification on P. ovale infections (p < 0.001). Misclassification of P. falciparum infections as MX was significantly higher compared to similar misclassification of P. malariae, P. ovale and P. vivax infections (p < 0.05). Residual post-training misclassification of P. falciparum infections as P. malariae was significantly lower compared to equivalent misclassification on P. ovale infections (p < 0.001). Misclassification of P. ovale infections as P. vivax was significantly higher compared to similar misclassifications on P. falciparum and P. malariae infections respectively (p < 0.001) and misclassification of P. vivax infections as P. ovale equally higher compared to equivalent misclassification on P. falciparum and P. malariae infections (p < 0.001)  Figure .Plasmodium infections [Publications by different groups have shown that laboratory personnel involved in microscopic diagnosis of malaria experience difficulties in differentiating fections -19. ThosPlasmodium infections based on species outcomes were most probably a result of guess work. Nonetheless, significant trends in misclassifications emerged in post-training assessments despite there being significant reductions in species identification errors. These shifts were majorly misclassification of P. falciparum infections as mixed infections, misclassification of P. ovale infections as P. vivax and misclassification of P. vivax infections as P. ovale.Lack of significant trends in pre-training misclassification of the four P. falciparum infections as mixed infections can be attributed to morphological variations in parasites forms. Whereas early trophozoites stages are smaller in size and have tiny nucleus and delicate cytoplasm, mature stages are relatively bigger in size, more compact and pigmented. This might have led some participants to believe non- falciparum species were also present on some of the blood films. Misclassification of P. ovale infections as P. vivax and vice versa might have been due to similarities in morphological characteristics such as cell inclusions i.e. James’ and Schüffner’s clefts, host cell enlargement and rounded gametocytes. These overlaps in morphological characteristics make differentiation of the two species extremely challenging even for experienced microscopists. A key feature commonly used in differentiating these two species is the presence of distinct amoeboid or fragmented forms and marked enlargement of the host cell in P. vivax infections.Misclassification of Plasmodium species. Most of the pre-training misclassifications were as a result of thick film examination. While thick films are useful in concentrating parasites for easy identification; the presence of multiple layers of red blood cells which are subsequently de-haemoglobinized during staining distorts essential morphological features needed for species differentiation. Unlike thick films, single layers of red blood cells at the tail of thin films and fixation in alcohol before staining helps in preserving species specific diagnostic details such as red blood cell shape and size, parasite morphology, pigment color and presence of cell inclusions which would otherwise be lost during thick film staining. Therefore, thin films are more reliable in species differentiation compared to thick films. Ideally, thick films should be used for presumptive identification of parasites and estimation of parasite counts whereas thin films should be used for definitive identification of species.Other than possession of requisite skills in morphological identification, thin films are equally important in enhancing the identification of Plasmodium infections and their respective multiple morphological characteristics.Post-training results did show significant reductions in misclassifications despite the residual errors reported. Occurrence of residual errors can be attributed to variations in morphological characteristics in various stages of parasite development within species and overlap in morphological characteristics between species. It is also important to note that proficiency in microscopic diagnosis of malaria is built over time, based on frequent exposure to different Plasmodium infections may be a common occurrence where non-falciparum species are involved. This can be attributed to lack of requisite skills in morphological characterization on the part of microscopists as well as morphological variations in parasite characteristics within and between species. Improving the skill sets of malaria microscopists through remedial training may reduce species misclassification errors associated with microscopy and ensure reliability of patient care and research results. Independent confirmation of species by expert microscopy in addition to molecular analysis of specimens in both patient care setting and research studies is highly recommended.Conventional light microscopy despite being the “gold standard” for malaria diagnosis is wrought with many challenges. These findings suggest misclassification of Plasmodium falciparum; PM: Plasmodium malariae; PO: Plasmodium ovale; PV: Plasmodium vivax; MX: Mixed infections.FN: False negative; PS: Positive without speciation; PF: All authors declared that they have no competing interest.PO designed the course, taught the courses, analyzed the data and drafted the manuscript. BO designed the course, facilitated financial and logistical support for courses conducted in Kenya and Ghana and assisted in drafting the manuscript. MA and JSO were the senior instructors of the courses. KL critiqued training methodology and results. CA designed the course, was responsible for logistics and taught the courses. DO was responsible for logistics and taught the courses. SO and FB facilitated financial and logistical support for courses conducted in Ghana. EW and JJ facilitated financial and logistical support for courses conducted in Kenya and assisted in drafting the manuscript. All authors read and approved the final manuscript.FN misclassifications within Plasmodium infections. Note: Values represent differences between comparisons together with the corresponding X2 statistic, X indicates redundant comparisons while † indicates significant differences.Click here for filePS misclassifications within Plasmodium infections. Note: Values represent differences between comparisons together with the corresponding X2 statistic, X indicates redundant comparisons while † indicates significant differences.Click here for fileSpecies misclassifications within Plasmodium infections. Note: Values represent differences between comparisons together with the corresponding X2 statistic, X indicates redundant comparisons while † indicates significant differences.Click here for fileMisclassifications between Plasmodium infections. Note: Values represent differences between comparisons together with the corresponding X2 statistic, X indicates redundant comparisons while † indicates significant differences.Click here for file


Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.Variations in cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.The results of this study indicate that  H. pylori is a microaerophilic, spiral shaped Gram-negative bacterium that chronically infects the gastric mucosa, H. pylori P12 [GeneBank:CP001217], H. pylori G27 [GenBank:CP001173], and H. pylori Shi470 [GeneBank:CP001072] sequences using the CLC DNA Workbench version 5.5[CagE and cag-PAI (empty-site) amplicon sizes were analysed by capillary gel electrophoresis only.M13-tagged sion 5.5. Sequencsion 5.5. CagE anP < 0.05. All statistical analyses presented here were significant according to Hosmer-Lemeshow (HL) goodness-of-fit test, with HL p values >0.05. In the logistic regression analysis and the GLM analysis, a 95% confidence interval including 1.0 was regarded as non-significant. Odds ratios with 95% confidence intervals (CI) were calculated to explore possible associations of individual genotypes to peptic ulcer or gastric atrophy. Age and sex were included as covariates. With regard to atrophy, data from duodenal biopsies were not included in the statistical analysis.Binary logistic regression analysis of data was performed using Minitab 15 software. Statistical significance was assumed at The authors declare that they have no competing interests.AK, AR, KB and HJM participated in the conception, design and data interpretation and drafting of the manuscript. AK, AR, MND performed the practical molecular biology procedures. KB collected and selected the biopsy specimens. All authors have been involved in drafting of the manuscript and approved the final version.cagA and vacA genotyping, including clinical data.Results from Click here for file


A number of studies have shown that proinflammatory cytokines have important roles in determining the development of microvascular diabetic complications, including nephropathy. Inflammatory biomarkers should be useful for diagnosis or monitoring of diabetic nephropathy. Mindin (spondin 2) is a member of the mindin-/F-spondin family of secreted extracellular matrix (ECM) proteins. Recent studies showed that mindin is essential for initiation of innate immune response and represents a unique pattern-recognition molecule in the ECM. Previously, we demonstrated that the levels of urinary mindin in patients with type 2 diabetes were higher than those in healthy individuals. We propose that urinary mindin is a potent biomarker for the development of diabetic nephropathy. Althougtic rats . Based otic rats . At prestic rats , 14. Inftic rats , 16, vastic rats , 18, montic rats , 20, and (TGF-β) , as wellGF-β) [α –24, are To survey the glomerular gene expression profile in type 2 diabetes, we performed a microarray analysis using isolated glomeruli from diabetic KK/Ta mice . We obseDrosophila M-spondin [in vitro [Mindin is a highly conserved extracellular matrix (ECM) protein that is abundantly expressed in the spleen and lymph nodes . It is a-spondin –37. Mind-spondin . Subsequ-spondin , 36. Mouin vitro . All memin vitro . The cryin vitro . Remarkain vitro  and as ain vitro , 28, 30.in vivo production of inflammatory cytokines during endotoxin-induced septic shock. Macrophages and mast cells from mindin-deficient mice show defective responses to a broad spectrum of microbial stimuli. Mindin recognizes LPS through its TSR domain [in vitro. Moreover, mindin induces nuclear-factor- (NF-) κB promoter activation in a TLR-9 mediated manner [As shown in R domain . Mindin R domain . Mice laR domain . Thus, mR domain , 40 . Moreover, mindin was reported as a key regulator of Rho GTPase expression. Signaling through integrins activates Rho GTPases [Podocytes cover the outer aspect of the glomerular basement membrane (GBM) and form the final barrier to protein loss . The pododocytes –46. Jia odocytes . We also GTPases . CytoskeIncreasing evidence indicates that inflammatory and immune response mechanisms may contribute significantly to the development and progression of diabetic nephropathy. Recent studies showed that mindin is essential for the initiation of immune response and represents a unique pattern-recognition molecule in the ECM. We focused on the role of mindin and examined the urinary secretion of mindin in mice and humans. Mindin could be an early biomarker of the progression of diabetic nephropathy.