Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae and M. hyorhinis infections may cause respiratory disease. Information regarding transcript structure and gene abundance provides valuable insight into gene function and regulation, which has not yet been analyzed on a genome-wide scale in these Mycoplasma species. In this study, we report the construction of transcriptome maps for M. hyopneumoniae, M. flocculare and M. hyorhinis, which represent data for conducting comparative studies on the transcriptional repertory. For each species, three cDNA libraries were generated, yielding averages of 415,265, 695,313 and 93,578 reads for M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively, with an average read length of 274 bp. The reads mapping showed that 92%, 98% and 96% of the predicted genes were transcribed in the M. hyopneumoniae, M. flocculare and M. hyorhinis genomes, respectively. Moreover, we showed that the majority of the genes are co-expressed, confirming the previously predicted transcription units. Finally, our data defined the RNA populations in detail, with the map transcript boundaries and transcription unit structures on a genome-wide scale.The swine respiratory ciliary epithelium is mainly colonized by  Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis are the most important species that have been identified in the swine respiratory system M. hyopneumoniae is the etiological agent of porcine enzootic pneumonia, whereas M. hyorhinis, in addition to being present in swine respiratory tracts, may cause swine serofibrinous to fibrinopurulent polyserositis and arthritis M. flocculare is also widespread in swine herds, but no disease has been associated with this species M. hyopneumoniae and M. flocculare are phylogenetically closely related species M. hyopneumoniae can adhere to the cilia of tracheal epithelial cells and cause damage. Although M. flocculare can also adhere to cilia, no important resulting damage has been observed, suggesting that M. hyopneumoniae and M. flocculare may possess different mechanisms of action involving the recognition of different receptor sites on the cilia M. hyorhinis to colonize other host sites M. hyopneumoniae, M. flocculare and M. hyorhinis possess small genomes with limited biosynthetic potentials. Six M. hyopneumoniae genome strains have been sequenced M. hyorhinis strains M. flocculare strain Similar to other mycoplasmas, M. hyopneumoniae 7448 genome M. hyopneumoniae genome by both in silico and in vitro approaches have suggested that the ORFs are continuously transcribed (cotranscribed) in large clusters (TUs). The authors have predicted that each TU is transcribed in the same direction with no intervening gene transcribed in the opposite orientation M. hyopneumoniae genome have also been reported in the M. flocculare and M. hyorhinis genomes Mycoplasma pneumoniaeAnalyses related to transcriptional unit (TU) organization, transcriptional regulation and promoter sequence composition have been performed on the Mycoplasma species that inhabit the swine respiratory tract and the limited knowledge available regarding mycoplasma transcriptional regulation, we aimed to analyze the whole transcriptomes of M. hyopneumoniae, M. flocculare and M. hyorhinis. In this work, we have sequenced, analyzed and constructed single nucleotide resolution transcriptome maps for the three species. A comparative analysis of the whole gene expression profiles between these swine respiratory mycoplasmas was also performed. Furthermore, the organizations of the genomes into large TUs (polycistronic mRNA), as previously predicted, were estimated and compared between the three species.Considering the importance of the three M. hyopneumoniae strain 7448, M. flocculare ATCC 27716 and M. hyorhinis ATCC 17981 were used in this study. The bacteria were grown in 25 ml Friis broth Total RNA was isolated with the RNeasy Mini Kit . For cell lysis, 0.7 ml of RNeasy Lysis Buffer (RLT buffer) in 0.134 M β-mercaptoethanol was used per cultivation flask. The purification was performed according to the manufacturer's instructions, with on-column DNase I digestion using the RNase-Free DNase Set  and a second round of treatment with DNase I . The absence of DNA in the RNA preparations was monitored by PCR. The extracted RNA was analyzed by gel electrophoresis and quantified using the Qubit system . RNA quality and integrity were determined by the evaluation of the RNA Integrity Number (RIN) using the Agilent 2100 Bioanalyzer . Values that were greater than or equal to 9.5 indicated sufficient quality.The ribosomal RNA (rRNA) depletion was performed using Terminator 5′-phosphate-dependent exonuclease , according to the manufacturer's instructions. Briefly, total RNA (10 µg) was combined with TEX 10X Reaction Buffer A, 20 U RiboGuard RNase Inhibitor, 10 U TEX (1 U/µg mRNA) and nuclease-free water in a final volume of 40 µl and incubated at 30°C for 60 minutes. The reaction was stopped with EDTA (0.5 M), and the mRNA was precipitated with 3 M sodium acetate and 2.5 volumes of ethanol. The mRNA was quantified using the Qubit system , and the effectiveness of the reaction was assessed by the Agilent 2100 Bioanalyzer . The absence of 16S and 23S rRNA in the posttreatment sample indicated a successful reaction.cDNA Rapid Library Preparation Method Manual GS FLX Titanium Series . After the library construction, the samples were quantified using the Qubit system , and average fragment sizes were determined using the Agilent 2100 Bioanalyzer . Three cDNA libraries for each strain were generated, totaling nine libraries, which were sequenced using the Roche/454 GS-FLX system. Following the analyses of the reads, each of the triplicate sets of libraries was pooled.The cDNA library preparation and pyrosequencing were performed using GS-FLX Titanium series reagents following the manufacturer's instructions . An equal amount of mRNA (200 ng) from each strain was used to synthesize the first and second strands of the cDNA, according to the http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for the read quality assessments, and the FASTX-Toolkit v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/) was employed for trimming. Reads were aligned with Bowtie2 v2.1.0 M. hyopneumoniae 7448, M. flocculare ATCC 27716 and M. hyorhinis HUB-1 genomic sequences were used as references, and the rRNA sequences were filtered. The results were parsed, and GenBank (gb) format files were generated for analysis with the Artemis Release 10.5.2 software package For the transcriptome assembly, Newbler v2.8 was used with the default parameters. The FastQC software (The Standard Flowgram Format (SFF) files of the sequence data generated in this study have been deposited in the Short Read Archive (SRA) database at NCBI under the accession number PRJNA255516.M. hyopneumoniae 7448 (NC_007332), M. flocculare ATCC 27716 (NZ_AFCG00000000.1) and M. hyorhinis HUB-1 (NC_014448.1).Expressed reads with coverage above background were mapped onto the annotated genes of M. hyopneumoniae 7448 M. flocculare and M. hyorhinis TUs were predicted The operon assessment was based on published experimental data from de novo assembly. The cDNA libraries were constructed with total mRNA from M. hyopneumoniae 7448, M. flocculare ATCC 27716 and M. hyorhinis ATCC 17981. The RNA preparations from the three species were obtained from bacteria grown under the same culture conditions. M. hyopneumoniae 7448, M. flocculare ATCC 27716 and M. hyorhinis ATCC 17981 genomes. The sequence coverage per base was subsequently plotted and visualized using the genome browsers Artemis and DNAplotter A total of 1,204,156 raw sequencing reads with an average length of 274 bp were generated using the Roche GS FLX Sequencing Platform. After cleaning and removing the adapters from each sequence in addition to low-quality sequences  and rRNA sequences, a total of 683,385 high-quality reads were obtained and used for the M. hyopneumoniae library allowed for the generation of 415,265 reads, of which approximately 94% mapped to the reference genome sequences of M. hyopneumoniae strain 7448. Most of the 695,313 reads (93%) from the M. flocculare library also mapped to the reference genome sequences of M. flocculare strain ATCC 27716. Similarly, 86% of the 93,578 reads from the M. hyorhinis library mapped to the reference genome sequences of M. hyorhinis strain HUB-1. The mapped M. hyopneumoniae, M. flocculare and M. hyorhinis genes represented 92%, 98% and 96% of the total predicted ORFs in these genomes, respectively  to genes from M. hyopneumoniae and other mycoplasma species. Three of these genes encoded hypothetical proteins, with the gene IDs MHP7448_0560, MHP7448_0709 and MHP7448_0138 in the M. hyopneumoniae 7448 genome, to which 127, 10 and 146 reads, respectively, were mapped in the M. flocculare transcriptome. Another novel identified gene encoded a lipoprotein with the gene ID MHP7448_0366 in M. hyopneumoniae, to which 31 reads were mapped in the M. flocculare transcriptome. A gene encoding a type III restriction-modification system: DNA methylase with the gene ID MHP7448_0386 in M. hyopneumoniae was also identified, to which 4 reads were mapped in the M. flocculare transcriptome.Genome annotation refinement is an important aspect of a transcriptome analysis. The transcriptome map of M. flocculare genome, the ftsZ gene is located in contig 04 and is positioned from nucleotides 8844–9491, which is located 647 bp downstream of a hypothetical gene (MF01093) in the opposite strand. However, the transcriptome map constructed in this study allowed for the identification of the incorrect annotation of the start codon of the ftsZ gene. The gene MF01093, with a highly expressed level   in contig 04. This observation was confirmed by BLASTX comparisons against homologous genes in other Mycoplasma sp.According to the current annotation of the 8.8) see , is in fM. hyorhinis allowed for the identification of two novel genes. The first encoded a hypothetical protein, HUB-1, which was positioned from nucleotides 721079–721572 (with 12 associated reads). BLAST comparisons indicated that this gene shared 98% identity  with MYM_0631, which is a hypothetical protein that is positioned from nucleotides 719192–719647 in the M. hyorhinis GDL-1 genome and totals 453 bp in size. The second non-annotated gene was rnpB, to which 7,876 reads were mapped. The BLASTX analysis showed that it shared homologies  with the genomes of other M. hyorhinis strains and other Mycoplasma species.The transcriptome map of M. hyorhinis HUB-1 genome. RNase P, which is encoded by the rnpB gene, is a ribonucleoprotein complex that removes 5′ leader sequences from tRNA precursors during tRNA biosynthesis. RNase P is present in all cells and subcellular compartments that synthesize tRNA, but catalytic activity by the RNA alone has been demonstrated only for bacterial RNase P RNA. Bacterial RNase P RNAs have been separated into two main structural classes. Type A (rnpA) is the most common structural class, and type B (rnpB) is found in low G+C gram-positive bacteria Mycoplasma sp. genomes typically contain both classes of RNase P.Additionally, in this work, we detected the endoribonuclease P (RNase P) ncRNA in the M. hyopneumoniae, M. flocculare and M. hyorhinis now allow for the detection of novel transcripts that have not been previously annotated in these genomes. Such transcripts include noncoding RNAs (ncRNAs), which were shown in other microorganisms to regulate important processes, such as pathogenesis, iron metabolism, and quorum sensing M. hyopneumoniae, M. flocculare and M. hyorhinis transcriptomes, respectively  ribonucleoprotein complex. The SRP RNA belongs to a group of small 4.5S RNAs that are found in bacteria, which direct the traffic of proteins within the cell and allow for their secretion. The SRP RNA, together with one or more SRP proteins, contribute to the binding and release of signal peptides In our M. hyopneumoniae transcriptome showed that 92% of the predicted genes possessed significant alignments with one or more sequences (rnpB), which encodes the RNA component of RNase P. Other genes that were also associated with high RPKM values included those encoding the cell division protein MraZ, S-adenosyl-methyltransferase, cell division protein FtsZ, p216 surface protein and hexosephosphate transport protein.The read distribution of the equences . A detaiM. flocculare genome revealed that 98% of the predicted genes significantly aligned to one or more sequences of the M. flocculare transcriptome , and another four hypothetical genes  with orthologs in the M. hyopneumoniae genome also presented with high RPKM values  presented with a significant amounts of transcripts   , similarM. hyopneumoniae genes encoding MHP7448_0198 (protein p97-copy 1) and MHP7448_0199 (protein p102-copy 1) are the only adhesin genes absent in the M. flocculare genome M. flocculare genome compared with those of the M. hyopneumoniae transcriptome (M. hyopneumoniae ortholog was MHP7448_0108 with RPKM  = 234.7) and MF00475  (the M. hyopneumoniae ortholog was MHP7448_0107 with RPKM  = 51.1). The finding of higher numbers of reads mapping in the M. flocculare adhesins compared with those of the M. hyopneumoniae orthologs was unexpected (see M. flocculare display inversions and rearrangements in contrast with those of M. hyopneumoniaeM. flocculare lacks the R1 domain M. hyopneumoniae adherence M. flocculare p97-like protein and its orthologs from M. hyopneumoniae (p97-like adhesin). Thus, even with the high read numbers of some of the adhesins that were observed in the current study, M. flocculare possesses a weak adhesion ability Previous analyses have shown that the orthologs of criptome , and evected see , but accM. flocculare, was elevated, as shown in M. hyopneumoniaeM. hyopneumoniae, compared to the M. flocculare ortholog with 278 reads (RPKM  = 72.5) , which has no ortholog in  = 72.5) . The P21M. hyorhinis was performed with the strain M. hyorhinis ATCC 17981. Because the genome of this strain was not available, the transcriptome assembly and mapping was based on the M. hyorhinis HUB-1 genome. The percentage of reads mapped (86%) was the lowest among the three mycoplasma cDNA libraries    also were aligned by expressive numbers of reads.The transcriptome analysis of ibraries . This recriptome , and app8,637.4) , followe1,456.1) . The genM. hyorhinis contains an exclusive variable lipoprotein (Vlp) system that constitutes its major coat proteins M. hyorhinis strains carry variable numbers of vlp genes M. hyorhinis HUB-1 is characterized by the presence of seven vlp genes displayed in the order 5-vlpD-vlpE-vlpF-insertion sequence (IS)-vlpG-vlpA-IS-vlpB-vlpC-3′ vlp genes were identified in the M. hyorhinis transcriptome map, the transcript numbers mapping to the different vlp genes were variable. vlpF, vlpE and vlpB were mapped by more reads compared with vlpG, vlpD and vlpC  and cell division (mraZ). Notably, the gene ftsZ, which encodes a cell division-related protein, is among the genes mapped with the largest RPKM amounts in the M. hyopneumoniae and M. flocculare transcriptomes (M. hyorhinis (RPKM  = 839.9). Most bacteria produce the tubulin homolog FtsZ ftsZ is non-essential for cell growth and in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery M. pneumoniae, very low levels of both ftsZ mRNA and FtsZ protein are present Abundant numbers of transcripts were identified in the three transcriptome maps that aligned to S-adenosyl-methyltransferase-associated genes, that participate in cell wall biosynthesis  . HSPs arM. hyopneumoniae, M. flocculare and M. hyorhinis has been recently described M. hyopneumoniae and M. flocculare. These two closely related mycoplasma species M. hyopneumoniae is considered to be pathogenic, and M. flocculare is considered to be a commensal species. This genome comparison showed many differences that may help to explain their differing behaviors. However, they were not able to identify specific virulence determinants that could explain the differences in their pathogenicities M. hyorhinis genome also shares significant numbers of genes with both M. hyopneumoniae and M. flocculare. The differences in M. hyorhinis gene composition seem to be related mainly to the ability of M. hyorhinis to colonize different hosts and sites.A comparative genome analysis between M. hyopneumoniae, M. flocculare and M. hyorhinis. The RNA-Seq data enabled us to determine the structures of the TUs on a genome-wide scale, which is important for the identification of co-expressed genes and for the understanding of the coordinated regulation of the mycoplasma transcriptome. Our results support previous predictions that the genes are preferably transcribed in polycistronic mRNA, forming TUs, in the mycoplasma genomes M. hyopneumoniae, M. flocculare and M. hyorhinis was described in silico prediction revealed that they are present in similar numbers in these three mycoplasmas genomes M. flocculare and M. hyorhinis similar to M. hyopneumoniaeM. hyopneumoniae, the overall gene distributions within the TUs in M. flocculare and M. hyorhinis are highly variable with respect to gene number and the functional categories of the encoded products in silico and in vitro approaches in M. hyopneumoniae in addition to the computationally predicted TUs in the M. flocculare and M. hyorhinis genomes were confirmed in the three transcriptomes analyzed here.We have constructed transcriptome maps of M. hyopneumoniae and M. hyorhinisM. flocculare transcriptome, all of the TUs predicted by the previous in silico analysis Over 80% of the TUs predicted for the three species were either confirmed or partially confirmed by our transcriptome analysis . Table 1M. hyopneumoniae 7448. The M. hyorhinis map has the largest mRNA contig mapped, with a size reaching of 13,000 bp (locations: 485595–498603 bp and 779997–788652 bp). M. pneumoniae transcriptome data showed a natural polarity with respect to the TU, in which the first gene of the TU exhibits the most prominent level of transcription, and levels progressively decreased for each successive gene in the TU The overlapping reads mapping to each reference genome generated a surprisingly large number of long contigs . Some exM. hyopneumoniae TUs are preceded by putative promoter sequences M. hyopneumoniae genomes M. hyopneumoniae TUs possess internal promoter sites, which is in agreement with the high variations in expression levels that were observed within the TUs. These findings are also similar to others that have been described in many bacterial species, including those in the M. pneumoniae transcriptome, which has been characterized by the frequent occurrence of polycistronic operons with alternative transcripts Bacillus subtilis transcriptome, in which 20% of the genes in polycistronic operons are transcribed from more than one promoter Halobacterium salinarum, 40% of the operons are expressed in condition-dependent manners Mycoplasmas are characterized by the presence of long TUs containing genes that encode highly functionally variable products. Moreover, all the Mycoplasma species that inhabit the respiratory tract of swine. We have analyzed the RNA populations present in M. hyopneumoniae, M. flocculare and M. hyorhinis in detail and used the data to map transcript boundaries and operon structures on a genome-wide scale. Moreover, we showed that almost all of the genes in these mycoplasma genomes are expressed at some basal level and that the majority of the genes are co-expressed, confirming the previously predicted TUs. Our results expand upon current knowledge regarding the coordination of transcription in swine respiratory mycoplasmas, in which genes are preferably transcribed in TUs. The determinations of all of the functional elements in the M. hyopneumoniae, M. flocculare and M. hyorhinis is a prerequisite for formulating holistic approaches to delineating the complex transcriptional regulation that occurs in these mycoplasma species.Overall, we provided comprehensive assessments of the transcriptomes of three important Figure S1Genome wide assessment. Circular plot of the reads mapping to the (A) M. hyopneumoniae 7448, (B) M. flocculare ATCC 27716 and (C) M. hyorhinis HUB-1 genomes. The outer circle (black) is marked in basepairs. The outermost circles represent CDS on the forward (outermost) and reverse (second outermost) strand (blue). Coding regions are illustrated by intermediate gray circle. The green inner circle represents the mapped sequence reads with a minimum quality score of 20. Visualization by DNAplotter.(TIF)Click here for additional data file.Figure S2Length distribution of contigs from M. hyopneumoniae (MHP), M. flocculare (MFL) and M. hyorhinis (MHR) transcriptome assembly.(JPG)Click here for additional data file.Table S1Transcriptome mapping from Mycoplasma hyopneumoniae.(XLSX)Click here for additional data file.Table S2Transcriptome mapping from Mycoplasma flocculare.(XLSX)Click here for additional data file.Table S3Transcriptome mapping from Mycoplasma hyrhinis.(XLSX)Click here for additional data file.Table S4Probably noncoding RNA detected in the three transcriptome studied.(XLSX)Click here for additional data file.Table S5Mycoplasma hyopneumoniae 7448 trancriptional units and cotranscription analysis.(XLS)Click here for additional data file.Table S6Mycoplasma flocculare trancriptional units and cotranscription analysis.(XLS)Click here for additional data file.Table S7Mycoplasma hyorhinis trancriptional units and cotranscription analysis.(XLS)Click here for additional data file.


Bifidobacterium adolescentis, which is one of many bacteria found in the human adult gut microbiota. In silico analysis of the genome sequences of eighteen B. adolescentis strains isolated from various environments, such as human milk, human feces and bovine rumen, revealed a high level of genetic variability, resulting in an open pan-genome. Compared to other bifidobacterial taxa such as Bifidobacterium bifidum and Bifidobacterium breve, the more extensive B. adolescentis pan-genome supports the hypothesis that the genetic arsenal of this taxon expanded so as to become more adaptable to the variable and changing ecological niche of the gut. These increased genetic capabilities are particularly evident for genes required for dietary glycan-breakdown.Bifidobacteria are members of the human gut microbiota, being numerically dominant in the colon of infants, while also being prevalent in the large intestine of adults. In this study, we determined and analyzed the pan-genome of  Bifidobacterium genus and for this reason designated the bifid shunt. Furthermore, genome-based analyses revealed that bifidobacteria follow varying genetic strategies to adapt to their particular ecological niche(s), many of which relate to the mammalian GIT5Bifidobacterium adolescentis appear to specifically colonize the gut of adult individuals and for this reason they represent a key bifidobacterial taxa of adult-associated bifidobacteriaB. adolescentis species has been carried out, revealing their extensive capabilities to metabolize diet-derived glycans, in particular starch and starch-related/derived poly- and oligo-saccharides, such as amylopectin, pullulan, maltotriose and maltodextrinBifidobacteria are a common component of the microbiota of the human gastrointestinal tract (GIT) and, in particular, they are amongst the first bacterial colonizers of the intestine of neonates12Bifidobacterium bifidumBifidobacterium breve111213915Pan-genomes of two other human gut bifidobacterial species, B. adolescentis is rather limited, being represented by six genome sequences, of which three that are still fragmented in many contigs68B. adolescentis strains that had been isolated from the adult gut or from rumen. Comparative genomic analyses of these sequences together with six other publicly available genome sequences of B. adolescentis species was performed. In addition, carbohydrate profiling of these strains was achieved involving various glycans including dietary- as well as host-derived glycans. Dietary changes are expected to impact on the ecological properties of the mammalian gut and thus on microbiota composition. The B. adolescentis taxon was shown to exhibit, through in silico and in vitro experiments, more extensive genetic flexibility and potential adaptive competitiveness to this highly variable ecological niche compared to other human bifidobacterial species.In contrast, the current genomic information available for B. adolescentis species, we isolated eight strains in addition to four strains  belonging to this taxon from different ecological niches, including human feces, human milk, and bovine rumen  48 (sub)species of the upertree , which dB. adolescentis strains were used to calculate the total gene repertoire encountered in this species. Thus, we evaluated the pan-genome and core-genome of the B. adolescentis taxon based on the clusters of orthologous genes (COGs) following a previously described methodB. adolescentis pan-genome that consists of 4448 COGs species of the genus Bifidobacterium. Furthermore, a varying number of unique genes, ranging from nine for B. adolescentis ATCC15703 to 251 for B. adolescentis 487B, were detected  values between B. adolescentis genomesB. adolescentis genomes belong to the same speciesB.adolescentis genome sequences was confirmed by comparative analyses using Dot-plot alignments . Furthermore, the analysis of this region revealed the presence of TUGs that appeared to have been acquired by horizontal gene transfer  as well as genes that are included in the core genome. These genes encode hypothetical proteins, putative phage proteins, several carbohydrate transport systems, enzymes that are involved in carbohydrate metabolism and the F1F0-ATPase cluster.Comparative genome analyses based on the 18 detected , represeignments . These aB. adolescentis taxon allowed the identification of three core genes that are uniquely present in this species , thus representing unique B. adolescentis core genes and encoding a Major Facilitator Superfamily (MFS) transporter, an acyltransferase and a hypothetical protein.Finally, the analysis of the core genome sequences of the B. adolescentis ATCC15703 to over 40% in the chromosomes of B. adolescentis 70B and B. adolescentis AD2-8, respectively  and an abundance of prophage-like elements. Additional putative mobile elements identified in the B. adolescentis genomes are represented by CRISPR loci and a Restriction Modification (R/M) system. Other variable regions were shown to include an EPS gene cluster and finally a type IVa pilus biosynthesis gene cluster (B. adolescentis 22LB. adolescentis LMG10734 (locus tags LMG10734_1251-LMG10734_1263), B. adolescentis LMG18897 (locus tags LMG18897_1585-LMG18897_1597), B. adolescentis 15O (locus tags 15O_1475-15O_1485), B. adolescentis 42B (locus tags 42B_1313-42B_1323), B. adolescentis 70B (locus tags 70B_1301-70B_1311), B. adolescentis 487B (locus tags 487_1645-487_1655), B. adolescentis AD2-8 (locus tags AD28_1414-AD28_1424) and B. adolescentis AL46-2 (locus tags AL46-2_1300-AL46-2_1310). The function of this cluster is unknown, yet in other microbes it has been linked to motility, conjugation, adherence and DNA uptakeB. adolescentis species is driven by various processes including conjugation, transformation, as well as phage-mediated transduction, which is consistent with previous publications29303132The mobilome represents the total number of genes that may have been acquired by HGT and its identification was performed using the software suite COLOMBO v3.8 implemented by SIGI-HMMectively . The pre cluster . The typB. adolescentis species, we performed an in silico prediction, in accordance to Carbohydrate-Active enZYmes Database (CAZy) databaseB. adolescentis genomes. This analysis revealed that the pan-genome of B. adolescentis contains genes encoding predicted carbohydrate-active enzymes, including 36 glycosyl hydrolase (GH) families, 12 glycosyl transferase (GT) families and four carbohydrate esterase (CE) families , which encompasses enzymes with predicted α-glucosidase, amylase, pullulanase, and cyclomaltodextrinase activities. In silico analyses also highlighted the presence of a number of GHs  that appear to be present in just a single strain of B. adolescentis species . In order to validate the above mentioned genomic-based analyses, we carried out growth experiments of B. adolescentis strains on 33 carbohydrates including both plant- and host-derived glycans, as unique carbon sources , ranging from dietary- as well as host-derived glycans82234families . Interes species , and the species , which a sources . As dispn starch .B. adolescentis strains assayed here was shown to be capable of utilizing mucin, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine or fucose, which indicates that the tested B. adolescentis strains possess limited metabolic capabilities to with regards to (monosaccharide components of) host-derived glycans  starch and starch-like polysaccharides, that are normally present in high abundance in the diet of adults.Notably, none of the  glycans . Such meB. adolescentis strains combined with six additional, publicly available B. adolescentis genomes. These results allowed the identification of an open pan-genome of the B. adolescentis species, which is in contrast to what has been observed for other human gut bifidobacterial species such as B. bifidum, B. breve, B. longum subsp. longum and B. animalis subsp. lactis91019B. adolescentis taxon exhibits greater genetic diversity compared to other human gut bifidobacterial (sub)species. Furthermore, the pan-genome of the B. adolescentis species is enriched in genes that are predicted to be involved in the metabolism of dietary, plant-derived glycans, in particular starch and starch-like oligo- and poly-saccharides, except in the case of two strains, B. adolescentis 703B and B. adolescentis JCM15918. In contrast, the B. adolescentis genomes do not appear to encode genes involved in the metabolism of host-derived glycans such as mucin and human milk oligosaccharides. These results supports the notion that the (human-associated) B. adolescentis species has evolved towards an ecological niche where plant-polysaccharides are present in high abundance, such as the large intestine of adult human beings. However, caution should be taken when drawing inferences based in silico predictions and in vitro experiments. Thus, future in vivo analyses performed in murine models or clinical trials will be needed in order to validate the results described in this study.The increasing number of publicly available bifidobacterial genomes offers the possibility to understand the evolution and the genomic diversity within this genus5B. adolescentis cultures were incubated in an anaerobic atmosphere  in a chamber  in de Man-Rogosa-Sharpe (MRS) (Scharlau Chemie) supplemented with 0.05% (wt/vol) L-cysteine hydrochloride and incubated at 37 °C for 16 h. Bacterial DNA was extracted as described previouslyThe strains used in this study are listed in All genomes used for this study were determined by GenProbio srl  using the MiSeq Illumina . Genomic libraries were constructed employing the TruSeq DNA PCR-Free LT Kit (Illumina) and using 2.5 μg of genomic DNA, which was fragmented with a Bioruptor NGS ultrasonicator  followed by size evaluation using Tape Station 2200 (Agilent Technologies). Library samples were loaded into a Flow Cell V3 600 cycles (Illumina) according to the technical support guide, and generated reads were depleted of adapter sequences, quality filtered and assembled through the MEGAnnotator pipelineB. adolescentis ATCC15703 as the reference genome (NCBI Accession Number: AP009256). Functional assignment was performed and manually edited based on similarity searches against the non-redundant protein database provided by the National Centre for Biotechnology Information. Artemishttp://www.expasy.ch/sprot//TrEMBL), PRIAM (http://priam.prabi.fr/), protein family , TIGRFAMs (http://www.jcvi.org/cms/research/projects/tigrfams/overview/), Interpro , Kyoto Encyclopedia of Genes and Genomes , and COG (http://www.ncbi.nlm.nih.gov/COG/) databases, in addition to BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Functional assignments were defined by manual processing of the combined results. Manual corrections to automated functional assignments were completed on an individual gene-by-gene basis as needed.Protein-encoding open reading frames (ORFs) were predicted using a combination of the methods Prodigalhttp://tubic.tju.edu.cn/GC-Profile/Ribosomal RNA genes were detected on the basis of BLASTN searches and annotated manually. Transfer RNA genes were identified using tRNAscan-SEB. adolescentis strain was searched for orthologues against the total proteome of B. adolescentis species, where orthology between two proteins was defined as the best bidirectional FASTA hits−4 and 30% identity over at least 80% of both protein sequences), and the resulting output was then clustered into protein families using MCL B. adolescentis genomes were classified. Protein families shared between all genomes, named core gene families, were defined by selecting the families that contained at least one single protein member for each genome.Each predicted proteome of a given http://tree.bio.ed.ac.uk/software/figtree/).Each set of orthologous proteins was aligned using CLUSTAL_WB. adolescentis genomes, a pan-genome tree was constructed. This tree was visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/).For all genomes used in this study, a pan-genome calculation was performed using PGAPPrediction and tree visualization of glycosyl hydrolase-encoding genes that had either been acquired or lost was performed with BlastGraphB. adolescentis strains were grown on semi-synthetic MRS medium supplemented with 0.5% (wt/vol) of a particular sugar and optical densities (OD at 600 nm) were recorded using a plate reader . The plate reader was run in discontinuous mode, with absorbance readings performed in 30 minutes intervals for 48 hours, where each reading was preceded by 30 s shaking at medium speed. Cultures were grown in biologically independent triplicates and the resulting growth data were expressed as the mean of these replicates. Carbohydrates were purchased from Sigma and Carbosynth .Fifteen SAMN04231308, SAMN04231309, SAMN04231310, SAMN04231311, SAMN04231312, SAMN04231313, SAMN04231314, SAMN04231315, SAMN04231316, SAMN04231317, SAMN04231318 and SAMN04231319).The sequences reported in this study have been deposited in the GenBank database .


Helicobacter pylori strain 7.13, a gerbil-adapted strain that causes gastric cancer in gerbils. Strain 7.13 is derived from clinical strain B128, isolated from a patient with a duodenal ulcer. This study reveals genes associated with the virulence of the strain.We report here the draft genome sequence of  Helicobacter pylori is a Gram-negative microaerophilic spiral bacterium. H. pylori is etiologically associated with chronic gastritis, peptic ulcer disease, and gastric cancer in humans. Several virulence factors play an important role in the pathogenesis of H. pylori-associated disease, including cytotoxin-associated gene A (cagA), vacuolating cytotoxin A (vacA), and induction by contact with epithelium gene A (iceA)  . Gerbil-al cells , 3.H. pylori strain 7.13 was sequenced using Illumina NextSeq 500 at Xcelris Labs. Ltd. The genome was sequenced with an average sequencing coverage of ~400×. The raw reads were quality filtered using Trimmomatic-0.30, and de novo assembly was carried out using ABySS, which generated 27 scaffolds (.The genome of cag pathogenicity island (cag PAI) and the vacA and iceA genes.The genome was annotated using the Rapid Annotations using Subsystems Technology (RAST) server . A totalH. pylori strain 7.13 was deposited in DDBJ/EMBL/GenBank with the accession no. LAQK00000000. The version of the strain described in this paper is the first version, LAQK01000000.The genome sequence of


Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors.An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium lavalense  is a recently described bacterial species [ species . It is G species . Its pat Clostridium lavalense in human blood cultures.Herein, we present the first reported isolation ofAn 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. The patient had a prior medical history of bilateral hip prosthesis, high blood pressure, and hypercholesterolemia. In the months preceding his admission, he had developed a right-sided hip pain. On the day of admission, his family had found him lying on the floor, too weak to walk. His blood pressure was 94/22 mmHg, heart rate 64 beats/min, and temperature 38.9°C. The examination of his head, neck, heart, lungs, and abdomen did not reveal anything abnormal. He had no wound nor sore. The mobilization of his right hip showed a limited range of motion without pain.9/L , a normal hemoglobin level of 135 g/L , and platelet count of 155 × 109/L . The creatinine was at 151 μmol/L , aspartate aminotransferases (AST) were at 63 U/L , creatine phosphokinase (CPK) was at 669 U/L , C-reactive protein (CRP) was at 230 mg/L , and procalcitonin was at 1.1 ng/mL . Four sets of aerobic and anaerobic blood cultures were collected on two consecutive days.Laboratory analysis revealed leukocytosis with a white blood cell count of 15.3 × 10A preliminary diagnostic of septic arthritis of the patient's right hip was suspected and a computed tomography (CT) scan of the pelvis was ordered. It showed no asymmetry or fluid collection around the hip prosthesis. The chest X-ray was read as normal. The patient was kept under observation, without antibiotic therapy. Clostridium species. No other organism was isolated. A commercial anaerobic manual identification panel  identified the bacillus as Clostridium butyricum, with >99.9% of probability. However, the isolate could not be identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) , after a complete extraction. Both empirical antibiotherapy with intravenous piperacillin-tazobactam and the search for the source of infection were initiated. A CT scan of the abdomen and pelvis showed multiple stones in the gallbladder without cholecystitis and no ascites nor colonic masses. A transthoracic echocardiogram revealed an aortic valve sclerosis without vegetation. A colonoscopy revealed three benign polyps without any sign of colitis or cancer. The patient defervesced after four days of intravenous piperacillin-tazobactam. On the 6th day of antibiotherapy, he was switched to oral amoxicillin for eleven more days. He was discharged from the hospital still weak, using a walker. Four months later, he had recovered his strength and was able to walk with a cane. He had no relapse of fever nor worsening of his right hip pain.The four anaerobic blood culture bottles became positive after 29 hours of incubation. Their Gram stain revealed the presence of Gram-negative rods. The culture showed the bacillus to be anaerobic, Gram-positive, and sporulated, typical of a Clostridium lavalense type strain CCRI-9842T. To identify the taxonomic neighbors of the clinical isolate, the 16S rRNA gene sequences were used for an initial BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) against GenBank. Phylogenetic and molecular evolutionary analyses with closely related Clostridium species were performed on 1,344 nucleotides with MegAlign® version 10 .Due to the discordance in identification between the RapID ANA II System and MALDI-TOF MS, the bacterium was sent to the Laboratoire de Santé Publique du Québec (LSPQ) for further characterization. It was designated LSPQ-04253 and was identified by 16S rRNA sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI 3130xl Genetic Analyzer  [ C. lavalense CCRI-9842T and CCRI-9929, C. asparagiforme DSM15981T, C. bolteae WAL16351T, C. citroniae CCUG52203T, C. clostridioforme ATC2553T, C. aldenense CCUG52204T, and C. butyricum VPI3266T revealed that LSPQ-04253 clustered with the two C. lavalense strains and was distant from C. butyricum VPI3266T  and those ofVPI3266T . The antVPI3266T . vanB gene was found in isolate LSPQ-04253 as described in the type strain CCRI-9842T. The vanB gene is carried by a mobile genetic element (transposon Tn5382) in the type strain CCRI-9842T that could be missing in isolate LSPQ-04253.No C. lavalense strains had been reported worldwide, CCRI-9842T and CCRI-9929, both from human faeces [ C. lavalense in human blood cultures. This indicates that C. lavalense is a potential human pathogen.Prior to the present case report, threen faeces , and str C. lavalense in the faecal microbiota of patients with Crohn's disease [Clostridia are present in the microbiota of the intestinal tract, the female genital tract, and the oral mucosa of humans . A recen disease . Clostridium spp. account for only 10% of such anaerobic bacteremia [ Clostridium bacteremia include hemodialysis, intestinal malignancy, and inflammatory bowel disease [ C. lavalense LSPQ-04253 into the patient's bloodstream remains unknown. The patient responded well to an empiric antibiotherapy with piperacillin-tazobactam, followed by amoxicillin. Susceptibility testing later confirmed the bacterium susceptibility to piperacillin-tazobactam. This finding is in agreement with a recent Canadian study on antimicrobial susceptibility of anaerobic bacteria in Ontario between 2010 and 2011. It showed that all non-perfringens Clostridium isolates were susceptible to piperacillin-tazobactam [Anaerobic bacteremia is uncommon, accounting for 5% of all positive blood cultures . Clostricteremia . Risk fa disease . Our patzobactam . C. butyricum by RapID ANA II but could not be identified using MALDI-TOF MS. It was clearly identified as C. lavalense by 16S rRNA gene sequencing and phylogenetic studies. This suggests that the bacteria involved in other cases of isolation of C. butyricum may have been misidentified and were in fact C. lavalense. A study on the epidemiology of Clostridium species bacteremia in Calgary between 2000 and 2006 showed that C. butyricum was involved in 4 of 138 Clostridium bacteremia cases [ C. lavalense 16S rRNA gene sequence belongs to the Clostridium coccoides rRNA group, the rRNA cluster XIVa of the genus Clostridium [ C. bolteae, C. citroniae, C. clostridioforme, and C. aldenense. Whether clinical cases which reported these four bacteria all identified the correct Clostridium species or failed to identify C. lavalense is unknown. Our work emphasizes the role of 16S rRNA gene sequencing and phylogenetic analysis in the proper identification of unusual pathogens and the importance of updating databases of conventional and new identification methods such as the RapID ANA II System and MALDI-TOF MS, respectively. The addition of C. lavalense in these databases will help in the proper identification of this bacterium in clinical cases. Further studies will be needed to define the role of C. lavalense in human disease and the presence of potential virulence factors.The bacterium was initially identified asia cases . The C.


Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis  species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species.  Bacillus pumilus and Bacillus safensis represent one of the most significant and widespread terrestrial species within the Bacillus pumilus group B. pumilus isolates have also been involved in foodborne poisoning B. pumilus isolates we have realized the difficulty to their differentiation from closely related species based on phenotypic and biochemical characteristics and on 16S rRNA gene sequences gyrB (β-subunit of DNA gyrase) and rpoB (β-subunit of RNA polymerase) has proven to be useful for taxonomic resolution of closely related species, including Bacillus species. Nevertheless, are already recognized its implementation difficulties in the routine of microbiology laboratories Studying a collection of previously identified Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), is an accurate, fast, and affordable emerging technique that has been increasingly used for identification of bacteria at different taxonomical levels B. pumilus and B. safensis cells Farfour et al. m/z 7620. Nevertheless, there is a need for further studies in order to assess the validity of this discriminatory tool. Moreover, Lash et al. Using vegetative Bacillus spp., ribosomal proteins and small acid-soluble spore proteins (SASPs) have been reported as potential species-specific biomarkers Bacillus cereus group species B. pumilus group.In addition, the species-specific protein fingerprint constitutes a valuable approach for the assignment of species biomarkers, which usually comprise cell structure and housekeeping proteins B. safensis and B. pumilus isolates. Moreover, the tentative assignment of their species-specific protein biomarkers was also performed.In this work, through the implementation of a protocol that enhances proteins extraction B. pumilus and twenty-two B. safensis isolates that were previously identified by phenotypic and genotypic  methods were studied B. safensis FO-36bT, B. pumilus ATCC 7061T and ATCC 14884 were included , which were subsequently cultured on the same medium, under aerobic conditions, for 24 h at 37°C. Bacterial cells were harvested by transferring three full loops (ca. 30 µl) from each agar plate into 20 µl of sterile water and were resuspended by vortexing. Bacterial inactivation was carried out applying the modified trifluoroacetic acid (TFA)  inactivation protocol −1 at 4°C. Subsequently, the supernatant was 10-fold diluted with HPLC grade water , filtered throughout a 0.22 µm pore size filter (Millipore) and stored at −20°C until further analysis.For MALDI-TOF-MS analysis, soluble proteins were extracted from a pure single colony of m/z 2000–12000, due to the good reproducibility of the spectral profile in this interval. All the spectra were externally calibrated using a commercial mixture of angiotensin I, ACTH (adrenocorticotropic hormone) and insulin  and analyzed with the Data Explorer software .Snapshots of different protein composition were detected and acquired by a MALDI-TOF/TOF mass spectrometer , equipped with a 200-Hz frequency Nd:YAG laser, operating at a wavelength of 355 nm. Pulse ion extraction with a 1300 ns delay time was used for collecting spectra. Measurements were carried out in linear positive mode using an acceleration voltage of 19.4 kV (Grid 1), and a lens 1 voltage of 8 kV. Each spectrum was the accumulated sum of at least 2000 laser shots within the ion range at For MALDI-TOF-MS experiments, 2 µl of the filtrated microbial dilution were mixed with 2 µl of a 12-mg/ml α-cyano-4-hydroxycinnamic acid (CHCA)  solution, prepared in 100% ACN  and 0.3% TFA. 1 µl of the mixture was spotted onto a 123×81 mm stainless steel MALDI sample plate  and allowed to dry at room temperature. For each isolate, two biological replicates (obtained from two different agar plates) were carried out, and the mean spectra were considered for the analysis. Mass spectra were analyzed with the Data Explorer software . Ion masses were extracted from the raw experimental mass spectra that included all the ion peaks with a relative signal to noise (S/N) ratio intensity above 2.MALDI-TOF-MS spectra were mean-centred and analysed by partial least squares discriminant analysis (PLSDA) http://web.expasy.org/tagident/) using 1% mass error for the taxonomic selections B. pumilus, B. safensis and B. subtilis. No restrictions on protein isoelectric point were used. This tool allowed the identification of proteins based on the experimental masses acquired by mass spectrometry using the information available at the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL protein sequence databases. Moreover, ribosomal proteins of genome-sequenced type strains, including B. pumilus ATCC 7061T available in the database developed by Hotta et al. Intact protein masses derived from MS analysis were used to generate a pool of candidate’s proteins for the identification of specific markers. The selected distinct mass information was submitted to a web-based TagIdent software tool (B. pumilus and B. safensisB. pumilus and B. safensis by molecular markers sequencing (e.g. gyrB), a time demanding and expensive methodology, which is not readily available for routine laboratories, was recently demonstrated B. pumilus and B. safensis discrimination despite the importance of the MALDI-TOF-MS application in bacterial differentiation. Moreover, combining accurate MALDI-TOF mass ion signals with the information available at a protein sequence database, such as UniProt, species-specific candidate protein biomarkers can be tentatively assigned, supporting the interest of this methodology for species identification.One of the current challenges in bacterial taxonomy is to integrate timely and accurate typing methods for a meaningful identification of microorganisms Bacterial species identification using MALDI-TOF-MS is based on mass profiles obtained from whole bacterial cell suspensions considering proteins with low mass weight (less than 20 kDa). Additionally, for MS applications it is imperative to define a standardized protocol, including the establishment of rigorous sample preparation and cultivation conditions, including culture medium, temperature and time. Therefore, an adequate number of ion masses should be obtained to allow the identification and discrimination of closely related bacterial species. Moreover, the definition of strict parameters for spectral data acquisition is also required.B. cereus group members, using a combined TFA treatment, centrifugation and filtration steps, is of relevance in the case of Bacillus species, since it promoted an efficient extraction of soluble microbial proteins presented in the core spore and other morphological structures B. pumilus and B. safensis isolates, clonally diverse and collected from different terrestrial origins The protein enrichment protocol, reported by Lash et al. Bacillus species under study: (a) B. pumilus (5 spectra) and (b) B. safensis (22 spectra), in the ion range at m/z 2000 to 12000. In fact, MALDI-TOF-MS profile analysis revealed the presence of species-specific ion signals. The m/z values at 3060, 3608.5, 5271 and 6122 were consistently observed in B. pumilus isolates, while the ions having the m/z values at 3396, 5288, 5568, 6094 and 6413 were related to B. safensis. Moreover, B. pumilus and B. safensis isolates demonstrated common ions at m/z 3692.5, 3821.5, 4305.5, 5948.5, 6704, 6793.5 and 7415  that were subjected to an extraction procedure previously to the MS analysis, suggested the presence of a series of ions at m/z 3620, 5297, 6617 and 7237, which were specific for this species, when compared with other B. subtilis group members  and with members of Bacillus cereus group . In addition, the presence of this same series of ions at m/z 3620, 6617 and 7238 were also detected by Fernández-No et al. B. pumilus species-specific discriminatory, when compared with B. safensis. Nevertheless, a closer inspection of the mass spectra of B. pumilus ATCC 7061T and 14884, revealed the presence of ions having m/z values at 3621, 5290 and 6624, although the ion at m/z 7238 was not present. Indeed, the differences found in these B. pumilus profiles could be justified by the distinct growth culture medium used and the sample preparation procedure employed.Farfour et al. attempted unsuccessfully to discriminate between B. pumilus and B. safensis. Results revealed the presence of two groups of characteristic ion peaks, comprising B. pumilus  and B. safensis . The authors claimed the presence of the additional ion at m/z 7620 in the spectra profile of B. safensis as a species-specific biomarker, allowing the discrimination of these two species. Nonetheless, analysis of our B. safensis spectral data (n = 22) did not reveal the presence of this ion peak, probably because the obtained MALDI-TOF-MS profiles were from vegetative cells. Additionally, the spectral profile obtained from spores seems to be more laborious and insufficient to discriminate appropriately among these closely related species, since spectral data with few number of ion peaks were generated. For these reasons, it was not possible to compare our study with the existing ones as we have used different cultural conditions and sample preparation procedures.Finally, in other work, Dickinson et al. B. pumilus isolates are available in public databases, as the SpectraBank (http://www.spectrabank.org), namely the type strain ATCC 7061T and the reference strain ATCC 14884, and no B. safensis was yet included, which constrains its identification.On the other hand, the species identification is limited to the bacterial species spectrum presented in a specific MS database. Therefore, bacterial identification is only possible inside the frame of bacterial reference spectrum of the database used. Indeed, few well-characterized B. pumilus and B. safensis were clearly discriminated by a combined MALDI-TOF-MS and chemometric approach. The score plot generated by PLSDA of mass spectra of all isolates tested exhibited two individualized clusters, each one enclosing isolates belonging to a particular spectral group, which included reference and type strains (B. pumilus and (b) B. safensis. The PLSDA scores were also presented as a dendrogram corroborating the species discrimination into two distinct clusters (Bacillus species with 100% of sensitivity and specificity. strains  of (a) Bclusters . MoreoveBacillus spp., this characterization was only previously applied in B. cereus group species B. pumilus group, stressing its relevance for discrimination among close related species.Despite the recognized proficiency of chemometric tools for the analysis and identification of bacteria based on their fingerprint, in the case of The possibility of biomarkers identification is one of the most valuable aspects of the mass spectrometric-based identification techniques, being this approach successfully applied to different bacterial species B. pumilus and B. safensis represented the first consistent evidence of the relation between these ion m/z signals and the specific candidate protein sequences, which were presented in B. pumilus genomes with numerous proteins defined as unknown were available in UniProtKB/Swiss-Prot and UniProtKB/TrEMBL (B. pumilus SAFR-032 and ATCC 7061T) and no B. safensis were deposited, which hindered the candidate biomarkers identification. Therefore, this type of assignments can only be tentatively used to establish potential connections between protein sequences and ion m/z signals, and thus, should be prudently interpreted, although previously successfully applied in B. cereus group members The assignment of 17 mass signals diagnostic ions formed in the MALDI-TOF-MS of B. pumilus and B. safensis specific biomarkers, associated with a series of ions at m/z 4305.5, 5948.5, 6793.5 and 7415, which was attributed to the 50S ribosomal subunits proteins, respectively, L36, L33, L28 and L35 of B. pumilus SAFR-032  and/or the transcriptional regulator – SlrA of B. subtilis subsp. subtilis str 168. Additionally, the characteristic ions at m/z 3060 and 3608.5 were not possible to assign.The tentative assignment of B. safensis specific ions at m/z 5288, 5568 and 6413, potentially corresponding with two specific SASPs (SASP J and SASP P) and a 50S ribosomal subunit protein L32 were also found in B. pumilus SAFR-032. The remaining ion m/z peaks detected were not possible to designate. Therefore, the proposed characteristic biomarkers, which could be used to differentiate between B. pumilus and B. safensis are summarized in B. pumilus and B. safensis.Concerning Bacillus endospores, have been also proposed as biomarkers for rapid differentiation and identification of Bacillus spp. using mass spectrometry approaches In fact, ribosomal proteins and small, acid-soluble spore proteins (SASPs) have been suggested to be responsible for many ion masses detected by MALDI-TOF-MS profiles B. pumilus and B. safensis biomarkers. A more detailed analysis could be carried out with MS/MS peptide fragmentation of the specific proteins assigned and subsequent comparison in protein databases or even with MS/MS peptide de novo sequencing. Nevertheless, within the context of the present work, which aimed to establish a MALDI-TOF-MS fingerprint classification for B. pumilus and B. safensis, these results may be beneficial and improve further accuracy of MS-based detection methods in identifying these species.Our results also suggested that ribosomal and spore proteins constituted most of the B. pumilus and B. safensis, allowing its rapid identification. These high throughput approaches should be promptly considered for Bacillus species identification due to the inaccuracy of conventional techniques in the identification of closely related species of this genus. In this sense, it is imperative to standardize a sample preparation protocol, which should include a protein extraction and enrichment step, to provide informative and reproducible mass spectra. Moreover, tentative assignment of B. pumilus and B. safensis protein biomarkers suggested that most of them are ribosomal and spore proteins.MALDI-TOF-MS profiles combined with chemometric analysis (PLSDA) proved to be valuable tools for discrimination of


T. cruzi infection, tegumentary and visceral leishmaniasis, malaria, and toxoplasmosis, both in patients and in animal models, which promote the understanding of immunopathological/immunoprotective parameters in these diseases.Protozoan infection is the cause of diseases of high morbidity and mortality. Most are non-self-limiting chronic infections and neglected diseases; emergent antimicrobial-resistant strains pose a substantial problem; for many of them treatment either is highly toxic or has limited effectiveness. Vaccine development is still a formidable task and there is no licensed vaccine for human protozoan infection. The key to the control of protozoan infection is the understanding of the host immune response to protozoan parasites, which will guide the development of effective vaccines and immunotherapeutic agents. In this special issue, there are important studies on the immunology of T. cruzi mucin (GPI-mucins) on human immune cells. It was observed that IL-12 production by GPI-mucins was dependent on IFN-γ and CD40-CD40L interactions. F. C. Dias and colleagues investigated the HLA-G expression in tissues and HLA-G 147 3′ UTR polymorphic site typing in patients presenting Chagas disease and the role of the mouse functional homolog in T. cruzi infection. F. Vorraro and colleagues studied the susceptibility to T. cruzi of mouse strains previously selected based on inflammatory and antibody responses to complex antigens. Q. Miao and M. Ndao review the potential impact of T. cruzi infection on the status of host lipid metabolism. The role of lipid mediators in T. cruzi infection is explored in two research articles. A. M. C. Canavaci and colleagues revisit the pathological role of 5-lipoxygenase in the acute infection of mice and A. D. Malvezi and coworkers investigate the role of cyclooxygenase and lipoxins in macrophage invasion by T. cruzi. In mouse models of chronic myocardial disease by T. cruzi, I. R. Pereira and colleagues show that treatment with infliximab, an anti-human TNF monoclonal antibody, reduced the frequency of mice with arrhythmias and cardiac fibrosis, while M. B. de Cuba and coworkers show the beneficial effect of cholinergic stimulation in reducing inflammation and fibrosis in the heart. Importantly, A. F. Araújo and coworkers demonstrate the beneficial effects of prophylactic vaccination using T. cruzi genes against acute and chronic pathologies caused by myotropic strains of the parasite. W. H. K. Cabrera and colleagues have characterized mouse lines selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction and for high (HIII) or low (LIII) antibody (Ab) responses to complex antigens. Resistance to T. cruzi infection was found in female and in high responder lines AIRmax and HIII. It was correlated with enhanced production of IFN-γ and nitric oxide production by peritoneal and lymph node cells, in HIII males and females. Moreover, an Ab production QTL marker mapping to mouse chromosome 1 significantly cosegregated with survival after acute T. cruzi infection.The reviews by J. M. Álvarez and colleagues and by E. Cunha-Neto and C. Chevillard cover fundamental findings, mechanisms, and questions concerning the immune response and pathology of mouse and human disease. In this line, L. G. Nogueira and colleagues characterize the myocardial expression of transcriptional factors involved in effector CD4+ T cell differentiation and the characteristic cytokines produced by the distinct lymphocyte subsets and demonstrate a profound predominance of local Th1 response. L. C. J. Abel and coworkers characterized the proinflammatory effects of glicophosphatidyl inositol-anchored Leishmania field, M. F. Lopes and colleagues here review the interface between Leishmania and phagocytes, cells that are both targets and effector of the anti-Leishmania immune response. The study by E. Svensjö and colleagues suggests that a serine peptidase (ISP-2) from L. major regulates macrophage phagocytosis by inhibiting the pericellular release of proinflammatory kinins from surface bound kininogens. C. M. V. Vendrame and coworkers have shown that insulin-like growth factor- (IGF-) I decreased nitric oxide production but increased arginase expression and activity, which lead to increased Leishmania parasitism. However, IGF-I did not result in altered cytokine levels. Moreover, stimulation with IGF-I induced phosphatidylserine exposure on amastigotes led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. M. D. T. Carvalho and colleagues investigated risk markers of L. infantum visceral leishmaniasis and found that lipoprotein and triglyceride levels are risk factors for development of visceral leishmaniasis. They also found that genetic polymorphisms at the Lpl and Pparγ were associated with differential levels of lipoproteins and thus related to the disease outcome. I. Naouar and coworkers investigated granzyme-positive CD4+ T cells activated by L. major antigens, in Leishmania-healed subjects. They found that even CD4+CD25+CD127dim/− Tregs expressed granzyme after exposure to Leishmania antigens.In the Plasmodium yoelii in MHC-I-Kd-Tg mice is mediated by CS protein-specific, Kd-restricted CD8+ T cells. E. S. Fernandes and colleagues studied the effect of TRPV1 antagonism by capsazepine during mouse infection with Plasmodium berghei ANKA. Their result indicated that there was a modulation of the innate immune response in mice infected but it did not affect parasitemia. Two other studies dealt with malaria pathogenesis. L. S. Ortolan and coworkers described new predictive criteria to study the pathogenesis of malaria-2 associated ali/ards in mice. This method for accurately identifying mice suffering from ALI/ARDS before death will allow the use of this model to study the pathogenesis of this disease. Finally, J. C. Sánchez-Arcila and colleagues evaluated whether intestinal parasites coinfection would alter plasma cytokines profile elicited in acute malaria in subjects from endemic area of Brazil. They concluded that the infection with intestinal parasites (mainly protozoan) does not significantly modify the pattern of cytokine production in individuals infected with P. falciparum and P. vivax.A number of manuscripts published in this issue provide important new knowledge in the important field of malaria. A review from H. Zheng and colleagues highlighted important aspects of preerythrocytic stages biology and the immune evasion strategies of malaria parasites. The unraveling of these escape mechanisms may aid the development of a vaccine against malaria. In this same direction, the manuscript by the group of J. Huang and colleagues generated a new mouse model to study the immunity to these preerythrocytic stages. This new model employed transgenic MHC-I Kd mice. The results of their studies indicate that protective antimalaria immunity induced by radiation-attenuated sporozoites of T. gondii and identified Th17 (CD4+) and Tc17 (CD8+) cells in toxoplasma-seronegative and toxoplasma-seropositive parturient and nonpregnant women. They observed a lower level of IL-17-expressing CD4+ and CD8+ T lymphocytes in cultures of cells from seronegative and seropositive parturient and nonpregnant women that were stimulated with tachyzoites. It has been shown that the cytokine pattern and IL-17-expressing CD4+ and CD8+ T lymphocytes may have important roles in the inflammatory response to T. gondii, thus contributing to the maintenance of pregnancy and control of parasite invasion and replication. A. S. Machado and coworkers studied immunological and hematological biomarkers of ocular congenital toxoplasmosis in infants, finding differential immune parameter networks in each clinical group .Finally, A. S. Machado and colleagues investigated the immunopathogenesis of toxoplasmosis in parturients and nonpregnant women. They measured the synthesis of Th1 and Th2 cytokines by mononuclear cells after culture with liveEdecio Cunha-NetoEdecio Cunha-NetoChristophe ChevillardChristophe ChevillardMauricio Martins RodriguesMauricio Martins RodriguesMarcelo T. BozzaMarcelo T. Bozza