Mutans streptococci are a group of bacteria significantly contributing to tooth decay. Their genetic variability is however still not well understood.S. mutans isolates of different origins, one isolate of S. sobrinus (DSM 20742) and one isolate of S. ratti (DSM 20564) were sequenced and comparatively analyzed. Genome alignment revealed a mosaic-like structure of genome arrangement. Genes related to pathogenicity are found to have high variations among the strains, whereas genes for oxidative stress resistance are well conserved, indicating the importance of this trait in the dental biofilm community. Analysis of genome-scale metabolic networks revealed significant differences in 42 pathways. A striking dissimilarity is the unique presence of two lactate oxidases in S. sobrinus DSM 20742, probably indicating an unusual capability of this strain in producing H2O2 and expanding its ecological niche. In addition, lactate oxidases may form with other enzymes a novel energetic pathway in S. sobrinus DSM 20742 that can remedy its deficiency in citrate utilization pathway.Genomes of 6 clinical S. mutans genomes currently available including the strains sequenced in this study, we estimates the theoretical core genome size of S. mutans, and performed modeling of S. mutans pan-genome by applying different fitting models. An “open” pan-genome was inferred.Using 67 The comparative genome analyses revealed diversities in the mutans streptococci group, especially with respect to the virulence related genes and metabolic pathways. The results are helpful for better understanding the evolution and adaptive mechanisms of these oral pathogen microorganisms and for combating them. By attaus.html) . They ocus.html) ,5. Many us.html) . Due to pathogen . Later, S. mutans.S. mutans strains , one S. ratti strain (DSM 20564) and one S. sobrinus strain (DSM 20742) and performed cross-comparison with the genome contents of S. mutans UA159 and NN2025, focusing on issues that are highly related to pathogenicity. The core- and pan-genome of S. mutans was analyzed by including 67 currently available S. mutans genome sequences. By constructing and comparing the genome-scale metabolic networks, the diversities in sub-networks (pathways) are systematically revealed. The results should be helpful for understanding the evolution and pathogenicity, as well as for prevention and treatment, of these very common opportunistic pathogens.In this study, we determined the whole genome sequences of six S. mutans strains are more close to each other than to S. ratti and S. sobrinus. This is consistent with the results of the phylogenetic analysis, as visualized by the phylogenetic tree constructed based on 16S rRNA and core genes single-nucleotide polymorphisms (SNPs) information shown in Figure S. mutans isolates as well as S. ratti DSM 20564 and S. sobrinus DSM 20742 is summarized in Table S. mutans strains, namely UA159 and S. mutans NN2025. The average GC contents are in the range of low GC organisms [S. sobrinus DSM 20742 and the smallest one from S. mutans KK23 showing merely 5.7% differences. The total numbers of protein-coding sequences per genome are also similar among all the strains compared.As expected, the overall genomic features of the eight rganisms . The genS. mutans NN2025 and UA159 discovered a large genomic inversion across the replication axis and similar genomic variations were also confirmed among 95 clinical S. mutans isolates using long-PCR analysis [S. mutans strains are determined by genome alignment using the MAUVE software [Genome rearrangements have important effects on bacterial phenotypes and the evolution of bacterial genomes . A compasoftware . The resS. mutans genome sequences. Compared to UA159 and NN2025, the chromosome segment represented by LCB E is reversely inserted between the LCB G and H in the strain AC4446, and between the LCB L and M in the strain KK23. This segment is related to the genomic island “SMU.100-SMU.116” of S. mutans UA159 which mainly contains sorbitol phosphotransferase system (PTS), transposase and hypothetical proteins [S. mutans which harbors a nonribosomal peptide synthetase-polyketide synthase gene cluster responsible for the biosynthesis of pigments [S. mutans UA159, additional genomic islands were found to be present/absent in the mutans streptococci strains of this study [As shown in Figure is study   was estimated to be around 1,370 genes, which is slightly lower than the calculated core-genome size  using 67 genomes. Compared with other streptococcus species, the core-genome of S. mutans is at the same level to the core-genome of S. pyogenes , less than that of S. pneumoniae  and S. agalactiae  [et al. [S. mutans was determined as 1,490 by using 57 S. mutans genomes which is obviously different to the core genome size of S. mutans we estimated, although we included the 57 S. mutans genomes used by Cornejo et al. in our study. The difference can be caused by different reasons, such as difference in the correction step for core gene determination and, very likely, different methods and parameter settings used for determining orthologs. Apparently, we have used a more stringent process to determine orthologs which led to smaller core genome size of S. mutans estimated.The core-genome size of the 67 n et al. ,20 to fistrains) ,22,23. Hy = a + bxc, y = a − bln(x + c) and y = a × ex/b−  + c  for modeling the pan-genome of S. mutans, as shown in Figure y = a + bxc and y = a − bln(x + c) indicated an infinite pan-genome, while the fitting result of using y = a × ex/b−  + c resulted in a negative value of the parameter a, suggesting a finite pan-genome However, the last fitting shows obvious deviations to many of the data points. Especially, the deviations even become larger with increased genome numbers, indicating that this model is not suitable. The best fitting result obtained with the model y = a + bxc shows fittings to all the data points with very high confidence. Based on this model, the pan-genome of S. mutans is still “open” while 67 genomes were included, and the expected average new gene number with the addition of a new genome is estimated to be 15. The infinite pan-genome was first proposed by Tettelin et al. for S. agalactiae based on the use of 9 S. agalactiae genomes. The three regression models used in this study are all based on the assumption that contingency genes are independently sampled from the pan-genome with equal probability, except in the case of “specific/unique genes”, which are modeled as unique events that appear only once in the entire global population. Hogg et al. [S. pneumoniae genomes, the predicted number of new genes drops sharply to zero when the number of genomes exceeds 50. However, in the case of S. mutans we could not observe such sharp decrease of new gene number even after 67 genomes were included. In the study of Cornejo et al. [S. mutans, after they used a special “pseudogene cluster” identification process to exclude about 30% of the rare genes that are considered to be pseudogenes. However, they didn’t provide detailed parameters they obtained from fitting. Our modeling using the 67 S. mutans genomes by applying the model described above without any restrictions pointed to an infinite pan-genome of S. mutans. However, we would like to understand this predicted “infinite” pan-genome as follows: 1) a “pan-genome” should be considered as “dynamic” rather than “static”, which means the pan-genome content is changing during the evolution, it does not matter if its size is infinite or finite; 2) The change of a pan-genome content can be caused by the acquirement of new genes or by the loss of genes; 3) The actual pan-genome size can be more stable than the content of the pan-genome but can also change during evolution coupled with the change of the environment. Thus, without considering the “gene loss events”, it’s quite understandable to have a “growing” or “infinite” pan-genome as gene acquirement occurs no matter how slow it might be. Interestingly, Cornejo et al. found a high rate of LGT in S. mutans, where many genes were acquired from related streptococci and bacterial strains predominantly residing not only in the oral cavity, but also in the respiratory tract, the digestive tract, cattle, genitalia, in insect pathogens and in the environment in general [We applied three models, namely g et al.  proposedo et al. , they pr general . Such hiS. mutans. In the following sections, systems that are highly related to stress resistance and pathogenicity are presented and discussed. As all the following results are based on putative alleles/ortholog groups established by OrthoMCL, if not otherwise specified, the word “putative allele(s)/ortholog(s)” is omitted in the following text.The annotated protein sequences of all the genomes studied were cross-compared based on alleles/ortholog groups established by the program OrthoMCL . In totaS. mutans[S. mutans, ComX, an alternative sigma factor, drives the transcription of the so called ‘late-competence genes’ required for genetic transformation. ComX activity is modulated by the inputs from two types of signal pathways, namely the competence-stimulating peptide (CSP) dependent competence regulation system and CSP-independent competence regulation system. ComX and the ‘late-competence genes’ regulated by ComX as labeled by boldface in Table In a previous study we have systematically discussed the two-component signal transduction systems (TCSTSs) in the 10 mutans streptococci strains . ComDE, S. mutans. In S. mS. mutans combines the action of the two ortholog systems which are present as ComABCDE and BlpABCRH in S. pneumoniae and involved in competence regulation and bacteriocins regulation, respectively. It should be noticed that, ComAB have been primarily considered to be the transporter of ComC, the precursor of CSP. Later, ComAB have been renamed as NlmTE as they were found to function together as transporter of nonlantibiotic bacteriocins, while another gene pair CslAB was supposed to be the transporter of ComC [It has been reported that the ComABCDE system in  of ComC . However of ComC .S. mutans, the comC-encoded prepeptide of CSP has a leader sequence containing a conserved double glycine (GG), at which the leader sequence is cleaved during transporting by ComAB to generate the mature signal peptide (CSP-21) containing 21 amino acid residues [comC alleles are present only in the eight S. mutans strains, not in the S. sobrinus DSM 20742 and S. ratti DSM 20564. Multi-alignment of the ComC sequences shows clear variations among different S. mutans strains  downstream of ComE that directly activates ComX has been identified by Mashburn-Warren et al. ComR activates the expression of the ComS, which is secreted, processed, and internalized through the peptide transporter OppD. The processed peptide, designated XIP (for sigma X-inducing peptide), modulates the activity of ComR, which in turn activates the expression of ComX. Deletion of comR or comS gene completely abolished the competence in S. mutans[S. sobrinus DSM 20742 which lacks the ComSR-coding genes. This well explains the fact that despite the presence of comX and the ‘late-competence genes’ we were not able to obtain the genetic competence state of S. sobrinus DSM 20742 (see discussion later in the “Variability and specificity in metabolic pathways and network” part). It is also worth to mention that the putative ComS ortholog found in S. ratti DSM 20564 is quite different to those of S. mutans strains, as shown in Figure In S. mutans. In thiscomE from S. mutans, suggesting that the CSP-dependent regulation system is one of the several signaling pathways involved in ComX activation [et al. identified in S. mutans another regulatory system, designated BsrRM, that primarily regulates bacteriocin-related genes but also affects the HdrMR system and thus indirectly contributes to competence development [S. ratti DSM 20564 nor in S. sobrinus DSM 20742. Furthermore, the response regulator BrsR of the BsrRM system is also absent in S. ratti DSM 20564, whereas S. sobrinus DSM 20742 lacks the complete BsrRM system. It’s also worth to mention that a competence damage-inducible protein CinA, which is regulated via ComX and has been proven to be related to DNA damage, genetic transformation and cell survival [It has been reported that a basal level of competence remains (referred as CSP-independent competence) after the deletion of tivation . Indeed,tivation . Moreovetivation ,41. Receelopment . In thissurvival , is presS. ratti DSM 20564 and especially in S. sobrinus DSM 20742 are very different to those of the S. mutans strains.Taking together, both the CSP-dependent and CSP-independent competence regulation systems in http://www.ncbi.nlm.nih.gov) and Oralgen  databases, as well as by searching for related publications. The collected protein sequences, as listed in Additional file Bacteriocins are proteinaceous toxins produced by bacteria to kill or inhibit the growth of similar or closely related bacterial strain(s). Bacteriocins produced by mutans streptococci are named “mutacins”. As dental plaque, the dominating niche of mutans streptococci, is a multispecies biofilm community that harbors many microorganism species, mutans group strains have developed a variety of mutacins to inhibit the growth of competitors, such as mitis group streptococci -46. In tStreptococcus bacteriocins has been reported previously [S. mutans strains and S. ratti DSM 20564, S. sobrinus DSM 20742 does not possess any genes coding for mutacin-like proteins. Mutacin-SMB has been identified in S. mutans and S. ratti previously [S. ratti DSM 20564 comprising 7 genes, including the mutacin-coding genes smbA and smbB, as well as 5 mutacin-related genes . Lantibiotic mutacins, mutacin-I [S. mutans strains KK23 and NN2025. Mutacin-K8 is an ortholog of the bacteriocin Streptococcin A-FF22 identified in group-A streptococci [S. mutans strain K8 [S. mutans UA159, 5DC8 and KK21, with only genes responsible for the immunity (scnFEG) left behind. Orthologous genes coding for a part of the mutacin-K8 production system are also found in S. mutans AC4446, consisting of only scnFEG, scnT (coding a lantibiotic exporter) and a part of scnM (coding the lantibiotic synthetase). Since a gene encoding ISSmu2-type transposase is found to be located upstream of mutacin-K8 precursor genes, we infer that the variety of mutacin-K8 production system in S. mutans strains studied here is highly possible to be caused by transposase actions.Diversity of eviously ,58. The eviously ,48. In outacin-I , mutacinutacin-I  and mutautacin-I , are comtrain K8 . By carenlmA/B  was discovered first in S. mutans UA140 to be active against the mitis group streptococci [nlmA/B are found to be present in six of the S. mutans strains, including UA159, 5DC8, KK21, KK23, ATCC 25175 and NCTC 11060, but not in S. mutans NN2025 and AC4446, nor in S. ratti DSM 20564 and S. sobrinus DSM 20742. On the other hand, the immunity protein for mutacin-IV (SMU.152), is identified in all strains, consistent with the fact that no inhibition phenomenon has been observed yet among different mutans streptococci strains. A mutacin-IV like protein found before in the strain UA159 (SMU.283) is identified in all strains except for S. sobrinus DSM 20742.Mutacin-IV, nonlantibiotic bacteriocins coded by ptococci . In thiscipB (SMU.1914) is found, in addition to S. sobrinus DSM 20742, also absent in the S. mutans strains ATCC 15175 and NCTC 11060. There are two homologs of mutacin-V immunity protein in S. mutans UA159, namely CipI (SMU.925) and SMU.1913 [cipB, SMU.1913 cannot prevent CipB-caused cell lysis in S. mutans UA159, and the key immunity factor of mutacin-V has been supposed to be CipI (SMU.925) rather than SMU.1913 [S. sobrinus DSM 20742 possess at least one orthologous gene encoding one of the two mutacin-V immunity proteins. Based on the similarity scores S. mutans NN2025 does not have an ortholog of CipI (SMU.925), but it possesses an ortholog (GI|290579764) of SMU.1913, which is possibly co-transcribed with GI|290579764, the cipB ortholog in S. mutans NN2025. Furthermore, the only putative immunity protein D822_3349 in S. ratti DSM 20564 shows very close similarities to SMU.925 (61%) and SMU.1913 (56%) and is possibly co-transcribed with D822_03354, the CipB ortholog in S. ratti DSM 20564. From these results, we suppose that SMU.1913, which is co-transcribed with cipB (SMU.1914), might be the ancestor gene coding for the mutacin-V immunity factor. The additional copy, like SMU.925 in S. mutans UA159, might be generated by duplication action and evolved as the dominant immunity factor in some of the mutans streptococci strains.Mutacin-V, another nonlantibiotic peptide coded by SMU.1913 ,60. ThesS. ratti DSM 20564. Putative ComAB, which has been proved to be the transporter complex of mutacin IV in S. mutans[S. mutans KK23 and S. mutans ATCC 25175.Furthermore, a possible nonlantibiotic bacteriocin peptide (SMU.423) is found to be present in all strains except for S. mutans, are ideTo summarize, a differed distribution of mutacin/bacteriocin encoding genes accompanied with a high conservation of genes coding for mutacin-immunity proteins are revealed for the 10 mutans streptococci strains/species. The conservation of mutacin immunity proteins apparently plays an important role for the survival of mutans streptococci strains under a bacteriocin-rich environment.8 per chromosomal replication [Bacteria and other microorganisms that cause infections are remarkably resilient and can develop ways to survive drugs meant to kill or weaken them. Antibiotic resistance can be a result of horizontal gene transfer , and alslication . The antS. mutans species is known to be intrinsically resistant to bacitracins produced by Bacillus subtilis. We confirmed this by testing all the 10 strains with a bacitracin-E-test (data not shown). All strains including S. ratti DSM 20564 and S. sobrinus DSM 20742 had a minimum inhibitory concentration between 128 and >256 μg/l. In fact, this antibiotic is used to isolate mutans-streptococci from highly heterogeneous oral microflora. It has been reported that bceABRS  system, encoding a two component signal transduction system and an ABC-transporter, is required for bacitracin resistance in S. mutans[bceABRS system is found to be present in all strains. Furthermore, an ortholog of a putative bacitracin resistance protein UppP  is present in all strains. It has been proved that overexpression of UppP in Escherichia coli and Bacillus subtilis results in bacitracin resistance [The S. mutans,64. As esistance ,66. HoweS. mutans NN2025, are found to be also present in S. mutans 5DC8 and S. mutans KK21. A vancomycin b-type resistance-associated protein (D822_01634) is uniquely present in S. ratti DSM 20564, but our phenotypic testing showed as expected that S. ratti DSM 20564 is susceptible to vancomycin. Furthermore, several putative multidrug resistance-associated proteins  are found to be present in all strains.Two penicillin-binding proteins (SMU.75 and SMU.455) are identified in all strains, indicating that they are potentially all susceptible to penicillin. Phenotypically all strains were tested to be susceptible to penicillin (data not shown). On the other hand, all the strains possess orthologs of SMU.368c, SMU.400, SMU.1444c and SMU.1515, which are homologs to beta-lactamases (EC 3.5.2.6), as well as orthologs of two so called beta-lactam resistance factors . Thus, all the strains are potentially capable of resistance against beta-lactam antibiotics. Orthologs of macrolide-efflux transporter proteins, as coded by GI|290581182 and GI|290581181 in 2-, H2O2, HO·) or adaptation to oxidative stresses aerobes and facultative anaerobes have evolved efficient defense systems, comprising an array of antioxidant enzymes such as catalase, superoxide dismutase (SOD), Dps-like peroxide resistance protein, alkylhydroperoxide reductase (AhpCF), glutathione reductase, and thiol reductase, which have been identified in many bacterial species.For protection against reactive oxygen species , AhpF/AhpC system, Dpr, thioredoxin system and glutaredoxin system, as shown in Table Although the first genome sequence of S. mutans[SOD, which catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide, is an important antioxidant defense in nearly all cells exposed to oxygen . SOD is S. mutans.2O2 to their respective alcohol and/or H2O. Both AhpF and AhpC are present in all S. mutans strains of this study and in S. ratti DSM 20564, but are absent in S. sobrinus DSM 20742. The natural missing of AhpF and AhpC in S. sobrinus indicates that AhpF/AhpC system is not an essential peroxide tolerance system for some mutans streptococci species. While studying a ahpF and ahpC double deletion mutant of S. mutans, Higuchi et al. [dpr gene, which encodes a ferritin-like iron-binding protein involved in oxygen tolerance by limiting the nonenzymatic hydroxyl radical synthesis via iron-catalyzed ‘Fenton reaction’ in S. mutans. Their further studies on the biological function of dpr found that dpr gene from S. mutans chromosome was capable of complementing an alkyl hydroperoxide reductase-deficient mutant of E. coli, as well as complementing the defect in peroxidase activity caused by the deletion of ahpF/ahpC in S. mutans, indicating that dpr plays an indispensable role in oxygen tolerance of S. mutans[dpr gene in oxygen tolerance.The AhpF/AhpC system catalyzes the NADH-dependent reduction of organic hydroperoxides and/or Hi et al.  found thS. mutans,70. Dpr S. sobrinus DSM 20742, which lacks any ortholog of SMU1169c. An ortholog of a thiol peroxidase-coding gene (tpx) is identified in all strains.Thioredoxins are a class of small redox mediator proteins known to be present in all organisms. They are involved in many important biological processes, including redox signaling. Thioredoxins are kept in the reduced state by the flavor enzyme thioredoxin reductase in a NADPH-dependent reaction . They acStreptococcus agalactiae as a model, it has been discovered that in GSH-containing Gram-positive bacteria GSH synthesis is catalyzed by one bifunctional protein, γ-glutamylcysteine synthetase-glutathione synthetase (γ-GCS-GS), encoded by one gene, gshAB. Homologs of γ-GCS-GS have been identified in the genomes of 19 mostly studied Gram-positive bacteria, including S. mutans[S. mutans strains, namely UA159, 5DC8, KK21, KK23, ATCC 25175, and NCTC 11060, as well as S. ratti DSM 20564, possess two glutathione reductase orthologs (SMU.140 and SMU.838). This could possibly convey these strains certain advantages in the re-generation of GSH from GSSG, which in turn would be helpful for oxidative resistance.Glutaredoxins share many functions of thioredoxins but are reduced by glutathione  rather than by a specific reductase. This means that glutaredoxins are oxidized by their corresponding substrates, and reduced non-enzymatically by GSH . OxidizeS. mutans. All comS. mutans[In addition, 3′-phosphoadenosine-5′-phosphate phosphatase activity has recently been reported to be required for superoxide stress tolerance in S. mutans. PutativS. mutans strains are very close to each other, with UA159, NN2025, AC4446, 5DC8 and KK21 having almost exactly the same size, and the networks of KK23, ATCC 25175 and NCTC 11060 being merely about 2% larger. While the size of the metabolic network of S. ratti DSM 20564 is comparable to those of the S. mutans strains, the metabolic network of S. sobrinus with 833 reactions and 853 metabolites is the smallest one, which have 62 less reactions and 60 less metabolites compared to the largest one of S. mutans NCTC 11060 (895 reactions and 913 metabolites).In order to reveal the metabolic variability of the mutans streptococci systematically, we have reconstructed and analyzed the genome-scale metabolic networks of all the strains sequenced with the method proposed by Ma and Zeng  and an uS. mutans UA159 as reference, the presence and absence of reactions in each of the strains/species compared are discovered and mapped into sub-pathways based on the KEGG pathway classification (http://www.genome.jp/kegg/pathway.html). As a result, among the 416 sub-pathways defined in the KEGG pathway database 46 sub-pathways demonstrated certain variations between the strains/species, as summarized in Additional file Despite the comparable network sizes, however, all the strains possess or lack certain reactions/metabolites, as revealed by detailed comparative analyses. Using the metabolic network of A key feature of the oral environment is that the nutrients available to the oral bacteria are always fluctuating between abundance and famine associated with human diet. Thus, the ability to quickly acquire and metabolize carbohydrates to produce energy and precursors for biosynthesis is essential for the survival of all oral bacteria. Due to their key roles in carbohydrates metabolism and energy production, glycolysis/gluconeogenesis, TCA cycle and pyruvate metabolism pathways are generally considered to be highly conserved among these oral bacteria. Although mutans streptococci strains/species are closely related species as revealed by phylogenetic tree analysis in this study Figure , differeStreptococcus lack cytochrome oxidases required for energy-linked oxygen metabolism and energy (in the form of ATP) required for survival and growth are generated by substrate level phosphorylation in the glycolysis pathway [Lactobacillus plantarum JDM1 and lactate oxidase (D823_06595) with a similarity of 65% to ZP_09448656.1 (accession number) of Lactobacillus mali KCTC 3596, are found to be uniquely present in S. sobrinus DSM 20742. These two enzymes catalyze the reaction of L-Lactate + O2= > Pyruvate + H2O2 and/or D-Lactate + O2= > Pyruvate + H2O2. It has been reported that in S. pneumoniae concerted action of lactate oxidase and pyruvate oxidase forms a novel energy-generation pathway by converting lactate acid to acetic acid under aerobic growth conditions [S. sobrinus DSM 20742, the function of the lactate oxidases in S. sobrinus DSM 20742 should be different to that of S. pneumoniae. By a close examination we hypothesize that lactate oxidase, together with pyruvate dehydrogenase, phosphate acetyl transferase and acetate kinase, could form a novel energy production pathway to convert lactate acid to acetate and simultaneously produce one additional ATP, as depicted Figure S. sobrinus DSM 20742 could also play a role in consuming lactate to regulate pH, which would be an advantage for S. sobrinus DSM 20742 in resistance to acid stress. In addition, this pathway could replenish Acetyl-CoA, an important intermediate for the biosynthesis of fatty acids and amino acids. This is for the first time that such an energy production pathway is proposed in Streptococcus species. Furthermore, lactate oxidase and lactate dehydrogenase could form a local NAD+ regeneration system, which would be certainly advantageous to S. sobrinus DSM 20742 under aerobic growth conditions. Moreover, it is known that mutans group streptococci and the mitis group streptococci are competitors, with S. mutans producing mutacins to kill the mitis group streptococci and the mitis group streptococci in turn produce H2O2 to kill mutans group streptococci [S. sobrinus DSM 20742 has the potential ability of producing H2O2 to kill not only competitors  but also macrophages [S. sobrinus DSM 20742 was verified by PCR experiments as shown in Additional file S. sobrinus strain AC153 also harbors homologous genes of lactate oxidase, suggesting that lactate oxidase may be conserved and play an important role in S. sobrinus. In the effort to clarify the functionality of lactate oxidase we tried to knock out the two genes encoding the two enzymes by PCR ligation mutagenesis according to the method of Lau PC et al. (2002). We applied different transformation methods  but were failed to obtain the desired recombinants. Then, to find out if S. sobrinus DSM 20742 is able to enter genetic competence state at all, we tried to transform S. sobrinus with plasmids replicative in other Streptococcus spp. like pDL278 , which catalyzes the cleavage of citrate into oxaloacetate and acetate, and oxaloacetate decarboxylase (EC 4.1.1.3), catalyzing the irreversible decarboxylation of oxaloacetate to pyruvate and CO2, are not found in S. sobrinus DSM 20742, as shown in Figure S. sobrinus DSM 20742 might lacks the ability in anaerobic utilization of citrate as a substrate. The disadvantages of S. sobrinus DSM 20742 in citrate utilization could be offset by the novel energy production pathway from lactate to acetate proposed above.In contrast to the unique harboring of lactate oxidases in EC 4.1.3., which cS. ratti DSM 20564. Pyruvate-phosphate dikinase has been found in propionic acid bacteria [Acetobacter xylinum clearly indicate that the function of this enzyme under physiological conditions favors the process of gluconeogenesis [A putative pyruvate-phosphate dikinase (EC 2.7.9.1), which catalyzes the interconversion between PEP and pyruvate, is found to be uniquely present in bacteria . The larogenesis . Metabologenesis . Under gAll the 10 strains of this study possess similarly an incomplete TCA cycle and the primary role of the existing TCA enzymes is most likely the synthesis of amino acid precursors as has been reported previously ,88.S. mutans strains, one S. ratti strain and one S. sobrinus strain were sequenced, annotated and compared together with S. mutans UA159 and NN2025. Multiple genome alignment showed extensive genome rearrangement among the eight strains of S. mutans. The core-genome size of S. mutans was determined to be around 1,370 genes by including 67 S. mutans genomes. A possibly open pan-genome of S. mutans was inferred.In the present study, the genomes of 8 mutans streptococci strains, including six comC-like genes could be identified in S. ratti and S. sobrinus; putative ComC amino acid sequences of S. mutans show clear variations; ComS and ComR are absent in S. sobrinus which well explains the fact that we were not able obtain genetic competence state of S. sobrinus by experiment, even though the ComX and the downstream competence development genes are well reserved; furthermore, the response regulators of the HdrMR and BsrRM systems, which are known to be involved in competence development, are missing in both S. ratti and S. sobrinus.Systematic comparative analyses were focused on competence regulation, bacteriocin (mutacin) production, antibiotic resistance, oxidative stress resistance, as well as central carbon metabolism and energy production pathways. Most of these systems show remarkable differences between the strains, except for oxidative stress resistance systems which are found to be well conserved. CSP-dependent and independent competence regulation systems are highly diverse in mutans streptococci: no Variation in mutacin-encoding genes is accompanied with the conservation of mutacin immunity proteins, which indicates apparently important roles of the mutacin immunity proteins for the survival of these mutans streptococci in a bacteriocin rich environment. The presence of various antibiotic resistance factors, together with the open pan-genome inferred, implies that attention should be paid to the potential of mutans streptococci in the development of antibiotic resistance.S. mutans UA159 as reference. By identifying lactate oxidases to be uniquely present in S. sobrinus DSM 20742, we proposed for the first time a novel energy production pathway in S. sobrinus. Additional functions of the lactate oxidases in connection with the proposed energy production pathway are also discussed.The sizes of the genome-scale metabolic networks of the 10 strains are very close to each other. Comparative analysis of sub-pathways reveals that 46 sub-pathways of all 416 sub-pathways defined in KEGG pathway database show variation using In conclusion, the genomes of mutans streptococci display remarkable differences, especially among different species. We believe that the strain-specific information provided in this study should be helpful to understand the evolution and adaptive mechanisms of those oral pathogens.S. mutans 5DC8 was isolated from root caries by David Beighton ; serotype c strain S. mutans AC4446 was isolated from a proven case of infective endocarditis in Dillingen (Germany), serotype c strain S. mutans KK21 was isolated from enamel caries of an adult by Susanne Kneist , serotype c strain S. mutans KK23 was isolated from enamel caries of a child by Susanne Kneist , Serotype c, type strain S. mutans ATCC 25175 was isolated from carious dentine, serotype f strain S. mutans NCTC 11060 was isolated in Denmark from a patient’s blood, serotype b strain S. ratti DSM 20564(=ATCC 19645) was isolated from caries lesion in rat, and finally, serotype non-d & non-g strain S. sobrinus DSM 20742 (= ATCC 33478) was isolated from human dental plaque. Serotype c is over-represented because 70-80% of all S. mutans isolates are of this serotype. However, non-c serotypes seem to be associated with cardiovascular diseases and this is represented in our study by the serotype f strain. Besides S. mutans, S. sobrinus is considered as a relevant cariogenic species in human. The genome sequences of S. mutans UA159 and S. mutans NN2025 were genome sequenced previously and obtained from NCBI genome database .All the newly sequenced strains were described previously . BrieflyS. mutans UA159 using Maq and break down the low quality area to obtain a collection of long contigs. Finally, the long contigs were used to close partial gaps of the initial assembly to improve the assembly quality using Phrap. The first version genome annotations were performed using mauve [http://biosystem.bt1.tu-harburg.de/) established with PathwayTools [http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html) and the whole-genome shotgun sequences have been deposited at DDBJ/EMBL/GenBank under the accessions of AOBX00000000 (S. mutans 5DC8), AOBY00000000 (S. mutans KK21), AOBZ00000000 (S. mutans KK23), AOCA00000000 (S. mutans AC4446), AOCB00000000 (S. mutans ATCC 25175), AOCC00000000 (S. mutans NCTC 11060), AOCD00000000 (S. ratti DSM 20564) and AOCE00000000 (S. sobrinus DSM 20742). The present study used the new version deposited at DDBJ/EMBL/GenBank. As we found out that in the annotated results from PGAAP some coding genes are missing, we did manual curation based on blast searches using known coding nucleotide sequences, the location of the missing coding sequences are given in Additional file All of the strains were sequenced using the Solexa sequencing platform at the Helmholtz Center for Infection Research in Braunschweig, Germany. The “high-quality draft”  genome sng mauve ,93, tRNAng mauve  and Blasng mauve , and thewayTools ,96. ThiswayTools . During Multiple genome alignments were computed by using the progressive Mauve algorithm of the Mauve software  with defS. mutans draft genomes of this study and the previously released complete genomes of S. mutans UA159 and NN2025, 59 newly released S. mutans genomes (2 completed and 57 drafts) available in NCBI till April 2013 were also included in the core- and pan-genome analysis of S. mutans. The accessions of the 59 genomes are as follows:In addition to the 6 AGWE00000000, AHRB00000000, AHRC00000000, AHRD00000000, AHRE00000000, AHRF00000000, AHRG00000000, AHRH00000000, AHRI00000000, AHRJ00000000, AHRK00000000, AHRL00000000, AHRM00000000, AHRN00000000, AHRO00000000, AHRP00000000, AHRQ00000000, AHRR00000000, AHRS00000000, AHRT00000000, AHRU00000000, AHRV00000000, AHRW00000000, AHRX00000000, AHRY00000000, AHRZ00000000, AHSA00000000, AHSB00000000, AHSC00000000, AHSD00000000, AHSE00000000, AHSF00000000, AHSG00000000, AHSH00000000, AHSI00000000, AHSJ00000000, AHSK00000000, AHSL00000000, AHSM00000000, AHSN00000000, AHSO00000000, AHSP00000000, AHSQ00000000, AHSR00000000, AHSS00000000, AHST00000000, AHSU00000000, AHSV00000000, AHSW00000000, AHSX00000000, AHSY00000000, AHSZ00000000, AHTA00000000, AHTB00000000, AHTC00000000, AHTD00000000, AHTE00000000, CP003686, AP012336.et al. [Data pre-processing for the core and pan-genome analysis were performed using a self-written perl script  with provisional R numbers of R10001 (C00186 + C00007 = > C00022 + C00027) and R10002 (C00256 + C00007 = > C00022 + C00027). Compared to the reaction graph or the metabolite graph, wherein either reactions or metabolites  are shown in an interconnected way, the bipartite network is more comprehensible because, similar to the biochemistry textbook, both the reactions and metabolites are visualized at mean time. Seventy-six non-enzymatic automatic reactions were also considered for the network construction. The construction of sub-networks was based on the KEGG pathway classification (http://www.genome.jp/kegg/pathway.html) with slight modification of addition of reaction catalyzed by lactate oxidase into Glycolysis/Gluconeogenesis pathway (MAP00010) and Pyruvate metabolism pathway (MAP00620). The software Cytoscape [The bipartite metabolic networks were constructed based on the connection matrix of updated KEGG reactions database according to Stelzer and Zeng  with addytoscape  was usedS. sobrinus DSM 20742 as well as a second S. sobrinus strain (AC153) and from strains S. mutans UA159 as well as S. ratti DSM 20564 (as negative controls) was performed. The primers used were: 5′- GAGCAGGATAATTGACAGTC -3′ (forward primer) and 5′- ACTCAGTGACGAATCAGTT -3′ (reverse primer), which were designed by using Primer Premier http://www.premierbiosoft.com/primerdesign/index.html) and Vector NTI 9.0 (InforMax), respectively. Conditions for this conventional PCR were: 94°C, 2 min; followed by 32 cycles of 94°C for 30s; annealing temperature 48°C for 30s; and 72°C for 90s; final extension at 72°C for 5 min; length of amplicon 1,175 bp.To verify the unique presence of the lactate oxidase (consecutive) coding genes D823_06595 and D823_06598 respectively and to exclude the possibility of contamination with e. g. human DNA during the process of genome sequencing, PCR amplification (using one primer pair covering both genes) with newly isolated DNA from To clarify the functionality of the two lactate oxidases, namely D823_06598 (Llod) and D823_06595 (lod), PCR ligation mutagenesis according to the method of  using prS. sobrinus cells. The first step was the preparation of pre-competent cells of S. sobrinus applying the methods according to [S. mutans was additionally used to introduce genetic constructs and plasmids into S. sobrinus cells. In this approach two various concentrations of CSP were used: 02 and 1μM, respectively. Transformation of S. mutans was used as a parallel control. All these experiments were carried out at least three times.For transformation, two natural transformation methods were first used to assay and optimize the natural transformation of the rding to  and [101rding to  applyinget al. [Later, electroporation experiment was carried out according to the procedure described by LeBlanc et al. . Variouset al.  as well et al.  was also′-triphosphate; PEP: Phosphoenolpyruvate; HGT: Horizontal gene transfer; LGT: Lateral gene transfer; SNP: Single-nucleotide polymorphism; LCB: Locally collinear block; multi-MUMs: Multiple maximal-unique-matches; COG: Clusters of orthologous groups of proteins; CSP: Competence stimulating peptide; XIP: Sigma X-inducing peptide; ABC transporter: ATP-binding cassette transporter; SOD: Superoxide dismutase; NAD+: Nicotinamide adenine dinucleotide; NADP+: Nicotinamide adenine dinucleotide phosphate; GSH: L-γ-glutamyl-L-cysteinylglycine; GSSG: Oxidized glutathione; GCS: Glutamylcysteine synthetase; GS: Glutathione synthetase; GCS-GS: Glutamylcysteine synthetase-glutathione synthetase; CoA: Coenzyme A.PCR: Polymerase Chain Reaction; PTS: Phosphotransferase system; TCA: Tricarboxylic acid cycle; ATP: adenosine-5The authors declare that they have no competing interests.S. sobrinus and the knock-out of the two genes for lactate oxidase. GC and IWD contributed to the microbial aspects and valuable discussions. AZE conceived of and supervised the study and revised the manuscript. All authors read and approved the final manuscript.LS carried out the bioinformatics analysis and wrote the draft. WW participated in the conception and coordination of the study and contributed significantly to results analysis and drafting the manuscript. GC provided strains with a verified identity. GC, AR, MR and IWD performed the PCR verification experiments. HS did the experiments on genomic competence of Presence/absence of genes related to known S. mutans genomic islands of 10 mutans streptococci strains.Click here for fileCore gene list of S. mutans.Click here for fileLittle square linear fitting details of the core and pan genome models.Click here for filePredicted ortholog groups of 10 mutans streptococci strains using OrthoMCL.Click here for fileSequences of mutacins used for the identification of putative mutacins in 10 mutans streptococci strains.Click here for fileConstructed genome wide metabolic networks in Cytoscape (*.cyc) format.Click here for fileComparative analysis of the metabolic pathways in the different metabolic networks using S. mutans UA159 as reference. Absent and unique reaction numbers of metabolic networks in strains compared to S. mutans UA159. Absent and unique EC numbers of metabolic networks in strains compared to S. mutans UA159.Click here for filePCR verifications of the unique presence of the lactate oxidase genes in S. sobrinus DSM 20742.Click here for fileThe locations of missing genes in NCBI genome annotation results.Click here for fileThe perl script used for core- and pan-genome analysis in this study.Click here for file


By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples.Bacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites.DGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, The underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size. Candida species (0.8%), in tumor library while Prevotella (11.6%), Veillonella (9.9%), Granulicatella (3.9%), Escherichia coli (2.4%), Oribacterium (2.2%), Fusobacterium (1.9%), Actinomyces (1.4%), Megasphaera (1.4%), Afipia (1.2%) and Leptotrichia (1.0%) in non-tumor library. Among others, genera Capnocytophaga, Selenomonas and Leptothrix were exclusive to non-tumor (control) tissues and Eubacterium[[G-3], Campylobacter and Catonella, confined only to tumor tissues. FigureThe distribution of relative abundance of 40 representative genera in combined library . However, the richness estimators, Chao1 and ACE were higher in tumor library than in non-tumor library. Evenness was greater with non-tumor samples as compared to tumor samples suggesting less abundant species at tumor site. Good’s coverage of the combined library was ~98% suggesting that 2 additional phylotypes would be recognized if 100 more clones were screened. Individual-based rarefaction curves calculated using PAST for the two library sets showed asymptote curve  lymphomas[Bacteria have the capacity to penetrate and invade various epithelial cells colonizing and inducing inflammation which may plausibly associate to cancer progression,64. For ymphomas. Recentlymphomas. To the ymphomas,51,52. Tymphomas.In this study, the observed differences in DGGE profiles of inter- group, 22.73%–90.24% among non-tumor and tumor groups, and intra- group diversity, 34.88%–87.23% within non-tumor group and 41.46%–100% within tumor group, signified some underlying changes in bacterial colonization of the tissues. Thus, even slight differences in bacterial profile of non-tumor and tumor tissues seem significant as samples were procured from the same individual. It is not surprising that fingerprints showed no significant differences in mean total number of bands. DGGE is a semi-quantitative method and the band intensities are also influenced by 16S rRNA gene copy numbers or co-migration of two or more sequence types or combination of these,68. HoweFirmicutes were highly prevalent at tumor site as observed earlier[Johnsonella (p < 0.1). The bacterial species associated with tumor tissues were far more diverse than that previously shown by culture-dependent[This was further substantiated by the results of 16S clonal analysis showing relatively distinct bacterial affiliations at non-tumor and tumor sites of OSCC subjects.  earlier,38,40. Aependent,33-36 anAn interesting observation related to clonal analysis was that the sequences when matched with the two known databases, RDP and HOMD for highest similarity showed similar results up to genus level. But at species level, the uncultivable phylotypes detected were 3.83% and ~60% by HOMD and RDP respectively. This may be due to differences in basic structure of two databases. Unlike RDP, HOMD is a curated database with 626 species and phylotypes based on 98.5% similarity cutoffs of full 1540-base 16S rRNA sequences and each oral taxon assigned a specific number.Actinomyces sp. oral taxon 181, Streptococcus sp. oral taxon 071, P. histicola, P. pallens, Selenomonas sputigena, V. dispar and phylotype, Leptotrichia sp. oral taxon 215 present in non-tumor tissues are known putative representatives of predominant genera in healthy oral microbiome[Prevotella has earlier been associated with different types of endodontic infections[Leptotrichia an opportunistic pathogen with bacteremia or sepsis producing lactic acid as a major metabolic end product[Granulicatella adiacens which was highly prevalent in non-tumor group is also a known agent of endocarditis[S. intermedius was predominant in 70% of OSCC subjects at both non-tumor and tumor sites. S. parasangunis II and O. sinus were also present at both sites. Oribacterium species are weakly fermentative forming metabolic end products, acetic and lactic acid[S. anginosus detected at 4 non-tumor and 2 tumor sites has been reported earlier in OSCC specimens[Streptococcus anginosus group comprised of three species, S. anginosus, S. constellatus and S. intermedius and are normal flora in humans, these bacteria are pathogens associated strongly with abscess formation and with infection in multiple body sites[Eubacterium and closely related strains found in our study at tumor sites are major bacterial groups in oral lesions and play important role in infections of root canal and periodontal pockets and use proteins and peptides derived from tissues and blood as energy source[Atopobium, F. nucleatum ss. vincentii and Parvimonas have been associated with endodontic infections or periodontitis[Most of the cultivable bacteria, crobiome. Prevotefections and Leptcarditis. S. intetic acid. S. angipecimens,38 and specimens. The Strdy sites. Assachay source. Also, Adontitis,77,78. Tdontitis.Streptococci were more prevalent at tumor sites as also reported earlier[Streptococcus sp. oral taxon 058, Peptosteptococcus stomatis, S. salivarius, S. gordonii, G. haemolysans, G. morbillorum, J. ignava and S. parasanguinis I, to be associated with tumor site. Van Houte et al.[Streptococci which produced large amounts of acid (pH < 4.2 in broth) in both coronal caries and root-surface caries. Streptococci are saccharolytic producing short chain organic acid from carbohydrates, thus lowering the pH of their local environment[P. stomatis found in oral cavity is weakly saccharolytic and produces fermented products, acetic, butyric, isobutyric, isovaleric and isocaproic acids[Gemella species like any other commensal are opportunistic pathogens known to cause serious local and systemic infections mainly in immune-suppressed patients[J. ignava can be a predicted new pathogen not detected in earlier studies and known to be associated with gingivitis and periodontitis[ earlier,34,35,80e et al.,82 identironment and alsoic acids. These mic acids,86 and ppatients,87 were patients. J. ignadontitis.Studies have shown association of tooth loss or periodontal diseases and oral cancer-91. PeriEnterobacteriaceae and Tenericutes lead to increase in methylation of multidrug resistance gene1 (MDR1 gene) and bacterial-triggered inflammation that correlates with regional nodal metastases over adjacent normal mucosa[The involvement of infection-triggered inflammations has been estimated in the pathogenesis of approximately 15–20% of human tumors,94. Recel mucosa.Mager et al. demonstrJ. ignava not detected earlier in tumor samples by culture- dependent or independent methods. However, these studies were performed with limited sample size. Therefore, further investigation with larger sample size using high throughput sequencing would validate these findings and broaden our perspective on bacterial association and oral cancer.Together, the results indicate that certain bacterial species/phylotypes detected in this study may play a role in triggering chronic inflammation in oral cavity and possibly be associated at different stages of cancer. This maThe authors declare that they have no competing interests.SP participated in the design, implementation, analysis, interpretation of the results and writing the manuscript. XJ participated in implementation and analysis. YL participated in analysis of DGGE profiles. CE, RY and BS participated in collecting and providing the samples. XL participated in interpretation of the results and writing the manuscript. DS conceived of the study and participated in the design, implementation, analysis, interpretation of the results and writing the manuscript. All authors read and approved the final manuscript.Figure S1. Distribution of relative abundance of classes detected by HOMD and RDP in tissue samples from non-tumor and tumor sites of OSCC subjects.Click here for fileFigure S2. Distribution of relative abundance of order detected by HOMD and RDP in tissue samples from non-tumor and tumor sites of OSCC subjects.Click here for fileFigure S3. Distribution of relative abundance of families detected by HOMD and RDP in tissue samples from non-tumor and tumor sites of OSCC subjects.Click here for fileFigure S4. (a) Individual-based rarefaction; and (b) Rank abundance curves for bacterial species associated with non-tumor tissue and tumor tissue libraries.Click here for file


Helicobacter pylori (H. pylori) infection with gastric cancer in Gangetic West Bengal. This study was done over a period of five years (2006–2010). The patients residing in the Gangetic areas of West Bengal presenting with upper gastrointestinal symptoms underwent UGI endoscopy. Among gastric cancer patients, demographic characteristics, symptomatology, macroscopic and histologic lesions and H. pylori status were analyzed. At confidence level 95%, “Z” and “P” value were calculated to find significance. Among 23851 patients underwent UGI endoscopy, 14106 were males, 9745 females, 17889 Hindus and 5962 Muslims. Among 462 gastric cancer patients, Male : Female 2.7 : 1, Hindus : Muslim 3 : 1, abdominal pain, indigestion, and weight-loss were commonest presentations. Antrum was the commonest site whereas ulceroproliferative type was commonest type. H. pylori positivity was 80.89% in adenocarcinoma with statistically significant relation with intestinal type. In future, our target will be to modify risk factors; it will need further demographic studies and analysis, so that we can detect it earliest.There is marked geographical variation in the distribution and incidence of stomach cancer. We tried here to describe the pattern of relationships of age, sex, religion distribution, symptom profile, histological subtypes and  H. pylori infection, reduce the risk of gastric cancer [H. pylori infection.The incidence of gastric carcinoma has fallen dramatically in the last 50 years, but according to IARC-Globocan 2008, it is the third commonest cause of cancer death after lung and liver cancer in male and after breast and lung cancer in female in the world. It is still the 2nd and 4th most common cancer in males and females, respectively , 2. Intec cancer . This caWe started performing our original and honest study only after getting permission from the ethical committee. This work has been approved by the appropriate ethical committee of our Institution. This study was performed over a period of five years. The study population was from different districts on both sides of Ganges in West Bengal, India . The patients were first screened by the doctors in rural urban health centers, public and district hospitals, nursing homes, and chambers, and then they were sent to hospitals or diagnostic centers of respective districts having facility for upper gastrointestinal endoscopy for evaluation of symptoms related to upper gastrointestinal tract. After getting informed consent from the subjects, demographic data was collected in a proforma containing structured questionnaire from patients and/or patients' party. Then esophagogastroduodenoscopy was performed using 15% xylocaine spray as local anesthesia.After detecting the lesion, at least eight biopsies were taken from the suspected areas in each patient. Biopsy materials were fixed in ten percent formalin at room temperature and later on processed for histopathology.Helicobacter pylori status. For Helicobacter pylori testing, rapid urease testing was performed by using HP kit-TM manufactured by Allied Marketing Corporation, Kolkata, with sensitivity of 90% and specificity of 90%. Colour change from yellow to red within the time period of 15–20 minutes confirmed H. pylori.Multiple biopsies were also taken from normal looking mucosa adjacent to the growth for determining Helicobacter pylori status were analyzed.After getting histologically confirmed gastric cancer patients, their demographic characteristics , symptomatology, types of lesions (macroscopic and histologic), and (1)Z values  have been calculated. P value indicates the maximum probability for a given level of significance.For significance of percentages, (2)95% CI for difference of percentage:(3)Chi-square test has been used with two degrees of freedom for Statistical methods used as follows.n = 462), 340 were males and 122 were females (ratio 2.7 : 1). Religion distribution showed 310 Hindus and 152 Muslims (ratio 3 : 1). P < 0.05). Helicobacter pylori was positive in 80.09% of gastric carcinoma with the commonest incidence in intestinal subtype (89.1%) followed by diffuse type (85.2%).A total of 23851 patients underwent upper GI endoscopy of which 14106 were males and 9745 were females; 17889 were Hindus and 5962 were Muslims. A total of 462 cases were detected to have gastric malignancy. Demographic data for age, sex, and religion were tabulated in , vomiting (78%), dysphagia (24%), and weight loss (62%) were predominant symptoms pertaining gastric carcinoma . In a reP < 0.05). Recent study from Kerala in India showed that though predominant site of cancer was antral mucosa, there was a trend towards proximal shift. Cherian et al. showed no change in site specificity of carcinoma of stomach in South Indian population [Various reports revealed progressive increase in proximal stomach cancer and concomitant decline in distal stomach cancer in the western world , 15. Reppulation . Again Qpulation . Afridi pulation .P < 0.0001). Macroscopically, gastric cancer has been classified into 4 types: type one: polypoidal lesion, type two: fungating lesion, type three: ulcerated lesion, and type four: infiltrating lesion on the gastric wall or linitis plastica lesion. But there is considerable overlap between the above different types. Qurieshi et al. showed 35.5% ulceroproliferative, 26% proliferative, 31% ulcerative, and 7.4% infiltrative lesions during endoscopic procedure performed in Kashmiri patients . AnotherHelicobacter pylori infection and intestinal metaplasia, associated with chronic atrophic gastritis [Over the past half century, the histological classification of gastric carcinoma has been largely based on Lauren's criteria, which describes that gastric carcinoma is of two major subtypes: (1) intestinal subtype, an expansive epidemic type, corpus-dominant, more common in males, black, and elder age group most commonly associated with astritis , 18. Herastritis . It origastritis . Third castritis . Relativastritis . Regardiastritis . On the astritis . In contastritis , our stuastritis . In our Helicobacter pylori, a gm negative bacillus, colonizes in stomach mucosa and triggers the progressive sequences of gastric lesions from chronic gastritis, gastric atrophy, intestinal metaplasia, dysplasia, and finally gastric carcinogenesis [H. pylori infection [H. pylori infection is mainly acquired in childhood through oral ingestion, and infection may persist throughout the life [Helicobacter pylori with age; it was mainly due to birth cohort rather than late acquisition of infection [ogenesis . Studiesnfection , 24. H. the life . Its prethe life , 27. Thenfection .Helicobacter pylori and risk of gastric adenocarcinoma. The risk is 2.1 to 16.7 fold greater than that in seronegative individual [H. pylori on gastric cancer development may vary with anatomic sites. Meta-analysis of prospective cohort studies showed that H. pylori infection was associated with rise of noncardia gastric adenocarcinoma [H. pylori infections are asymptomatic. Virulent strain of H. pylori carrying cytotoxin associated gene A (cagA) is responsible for gastric cancer in distal sites following atrophic gastritis [H. pylori detected by CLO test and histology was 66% out of 50 cases of gastric cancer [H. pylori in 71.8% of gastric cancer patients and 20% among control group, showing significant (P < 0.001) difference. Among the patients with intestinal type gastric cancer, H. pylori was present in 86.96% of cases, 50% of diffuse type and poorly differentiated type harbored H. pylori infection [H. pylori positive [H. pylori positive, whereas among the patients with diffuse signet ring type gastric carcinoma, 29% were H. pylori positive [H. pylori and gastric cancer [H. pylori infection in adult population in India is 55%–92%, as compared to 44% and 55% in Chinese and Japanese population, respectively. In our study, 80.09% of gastric carcinoma was H. pylori positive, which was contradictory to the observation in developing countries by Miwa et al. and other Indian studies [H. pylori positive. Our observation of H. pylori positivity in case of intestinal type was similar to observation by Kabir et al. In comparison to other Indian studies, H. pylori positivity in case of gastric cancer in West Bengal was much higher.Several case control studies have shown significant association between dividual , 30. Effarcinoma . Most ofastritis , 33. In c cancer . Kabir enfection . In studpositive . A studypositive . Other Ic cancer –37. Sero studies . 89.1% oH. pylori infection. Macroscopically ulcerative type was detected as the commonest lesion. Future target for gastric cancer prevention is to modify the risk factors, and further demographic studies and analysis for future screening will be needed.In summary, in this study the male and female ratio was 2.7 : 1, religion trend Hindus : Muslim 3 : 1. Median age of Stomach cancer in Gangetic West Bengal, India, was mainly around 55 years of age. Abdominal pain indigestion and weight loss were the commonest symptoms of presentation. Anatomically antral mucosa was mostly commonly involved. Surprisingly Fundus was significantly involved in females. Histologically interstitial type was the most common subtype of adenocarcinoma and more commonly associated with


Gibberella fujikuroi species complex, the exact relationships among the so-called “African”, “Asian” and “American” Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades  were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex.The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi (“Asian” Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets.Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes  in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.The mitochondrial genomes of  The origin of mitochondria can be traced to about one billion years ago when an endosymbiotic association emerged between α-proteobacteria and the “proto eukaryotic” host cell ,2. SinceThe mt genome is considered to be an ideal region to study eukaryotic evolution . This isFusarium species in the Gibberella fujikuroi complex (GFC). The GFC represents a monophyletic assemblage of Fusarium species, the majority of which are of considerable agricultural, medical and veterinary importance , F. verticillioides[F. fujikuroi [GenBank accession number JX910420] represent circular molecules with sizes of respectively 67 106, 53 753 and 46 927 base pairs (bp), whereas those of F. graminearum, F. oxysporum and F. solani are 95 676, 34 476 bp and 62 978 bp, respectively   from single reads that were initially identified as being of mt origin following a whole-genome reference assembly to F. oxysporum and F. graminearum. All contigs were exported to BioEdit for manual assembly using the F. oxysporum and F. graminearum sequences as references. Finally, primer3 (http://primer3.sourceforge.net/) was used to design primers to amplify regions between non-overlapping contigs (two in F. fujikuroi and one in F. circinatum). These primers were also used for standard Sanger sequencing to fill the gaps and to complete the mt genomes of these two fungi.The mt DNA sequences were extracted from various whole genome sequence libraries. h Africa  and F. f Germany . FollowiI 19292)  and F. gm (PH-1) . AdditioF. circinatum and F. fujikuroi, mt genes were identified by similarity searches against the mt genes of F. oxysporum and F. graminearum using BioEdit and the CLC Genomics Workbench. Open reading frames (ORFs) were identified with ORF Finder using genetic code 4  [http://www.protocol-online.org. Genes encoding tRNAs were identified with tRNAscan-SE [For hondria)  and BLASAscan-SE , which iF. oxysporum and F. graminearum. Introns were characterized using RNAweasel  [http://www.ebi.ac.uk/Tools/pfa/iprscan). To test whether the presence of introns is conserved within species, primers flanking introns identified within the cox1 gene were designed and 8 additional isolates of F. circinatum and of F. verticillioies were screened for the presence or absence of introns.Introns were identified using comparisons of sequence alignments against the known mt genes of Aweasel) . Homing F. circinatum, F. fujikuroi as well as F. verticillioides, F. oxysporum, F. graminearum and F. solani were determined using the CLC Genomics Workbench. Physical maps and a BLAST comparison, which also included the F. oxysporum mt genome, were constructed with CGView Server . Intergenic inverted repeats for each genome were identified with Einverted EMBOSS [http://zlab.bu.edu/repfind) with default settings . The default parameters excluded one nucleotide and dinucleotide tandem repeats  and identified only repeats that are expected to occur by chance on average once in 10 000 bp. Finally, we searched within the intergenic regions of the mt genomes of F. solani, F. oxysporum and F. graminearum in order to determine whether they share any of the GFC repeat elements.Overall nucleotide sequence identities of d EMBOSS  and intehttp://align.bmr.kyushu-u.ac.jp/mafft/online/server/) [http://www.ibi.vu.nl/programs/pralinewww) were used to generate nucleotide and amino acid alignments respectively. Best-fit substitution models were determined with jModeltest [All phylogenetic analyses were based on maximum likelihood (ML) and were performed with PhyML version 2.4.3 . MAFFT vserver/) ,73 and Podeltest  and Protodeltest  for nuclHypocreales for which mt genome sequences are available. These were F. verticillioides (JN041210) [F. oxysporum  [F. graminearum (DQ364632) [F. solani (JN041209), Hypocrea jecorina (NC003388) [Metarhizium anisopliae (AY884128) [Lecanicillium muscarium (NC004514) [cox1, cox2, nad5 and nad6 utilized the GTR + G. The cox3 nucleotide dataset also utilized the GTR + I. The TIM1 and TIM2 [atp6, atp9, cob, nad3, nad4 and the nad1, nad4L datasets respectively, while TVM [nad2 and atp8. ML bootstrap confidence values were based on a 1000 replications. The likelihood of alternative tree topologies was tested using the Shimodaira-Hasegawa (SH) test in PAUP* version 10b [Phylogenetic analysis of the various mt genes also included sequences for other species in the N041210) , F. oxysY945289) ,41, F. gQ364632) , F. solaC003388) , Metarhiani JN0419, HypocrC004514) , while tC004514)  with equand TIM2  substituhile TVM  with G ahile TVM  were ression 10b .Saccharomyces species. For this we focused on Saccharomyces cerevisiae (NC_001224), S. pastorianus (NC_012145), S. castellii (NC_003920) and S. servazzii, (NC_004918), using Candida glabrata (NC_004691) as outgroup species. These species were recently used for phylogenetic comparisons by Gaillardin et al.[cob and cox1utilized the TVM + G, while TIM1, GTR and TPM1 [atp8, atp6 and cox2, respectively. ML bootstrap confidence values were based on a 1000 replications.In order to determine if the single-mt-gene-tree incongruence observed in this study are also observed in other fungal groups, we included additional phylogenetic analysis of the mt sequences of in et al.. For ML and TPM1  all withF. oxysporum, F. graminearum. However, due to the general lack of sequence homology outside the motif characteristic of the endonucleaese families [Phylogenetic analyses were used to determine relatedness among the identified HEGs encoded in the introns. These analyses also included sequences for the HEGs identified from families , only thThe authors declare that they have no competing interests.in silico and laboratory procedures. All authors have read and approved the final manuscript.All authors participated in the conception and design of the study, the interpretation of findings and drafting of the manuscript. GF performed all of the Fusarium circinatum, F. verticillioides and F. fujikuroi. Table S2. Inverted, direct and dispersed repeats identified in the mitochondrial genomes of Fusarium circinatum, F. verticillioides and F. fujikuroi.Table S3. Intron distribution, type, size, and endonuclease of F. circinatum, F. verticillioides, F. fujikuroi, F. oxysporum, F. graminearum and F. solani. Table S4. Comparison of the alternative trees using the SH test. Figure S1. Physical map and BLAST comparison of the mt genomes of F. circinatum against F. oxysporum, F. verticillioides and F. fujikuroi. Figure S2. Midpoint rooted maximum likelihood phylogenetic tree of the amino acid LAGLIDADG endonuclease domains identified within intron regions of F. circinatum, F. verticillioides, F. fujikuroi, F. oxysporum, F. graminearum and F. solani.Figure S3. Midpoint rooted maximum likelihood phylogenetic tree of the amino acid GIY-YIG endonuclease domains identified within intron regions of F. circinatum, F. verticillioides, F. fujikuroi, F. oxysporum, F. graminearum and F. solani. Figure S4. Maximum likelihood phylogenies for Fusarium based on mitochondrial protein-coding nucleotide sequences. Figure S5. Maximum likelihood phylogenies for Saccharomyces species based on mitochondrial protein-coding nucleotide sequences.Mitochondrial amino acid codon usage and tRNA anti-codon sequences for Click here for file


Salmonella enterica to contaminate and grow in plant environments remain poorly described. We examined the effect of feeding by phytophagous insects on survival of S. enterica on lettuce. Larger S. enterica populations were found on leaves infested with Macrosteles quadrilineatus. In contrast, pathogen populations among plants exposed to Frankliniella occidentalis or Myzus persicae were similar to those without insects. However, on plants infested with F. occidentalis, areas of the infested leaf with feeding damage sustained higher S. enterica populations than areas without damage. The spatial distribution of S. enterica cells on leaves infested with F. occidentalis may be altered resulting in higher populations in feeding lesions or survival may be different across a leaf dependent on local damage. Results suggest the possibility of some specificity with select insects and the persistence of S. enterica. Additionally, we demonstrated the potential for phytophagous insects to become contaminated with S. enterica from contaminated plant material. S. enterica was detected in approximately 50% of all M. quadrilineatus, F. occidentalis, and M. persicae after 24 h exposure to contaminated leaves. Particularly, 17% of F. occidentalis, the smallest of the insects tested, harbored more than 102 CFU/F. occidentalis. Our results show that phytophagous insects may influence the population dynamics of S. enterica in agricultural crops. This study provides evidence of a human bacterial pathogen interacting with phytophagous insect during plant infestation. Recently, most foodborne illness outbreaks of salmonellosis have been caused by consumption of contaminated fresh produce. Yet, the mechanisms that allow the human pathogen  Salmonella enterica is the number one cause of bacterial food-borne illness, and the incidence of infection has not declined over the past 15 years, and instead, has increased slightly since mid-2000s [The frequency and severity of produce-related foodborne illness outbreaks have increased in the last few decades ,2. Althoid-2000s ,4. RecenS. enterica on leaves is rare and populations tend to decline steadily overtime [ Human pathogens experience harsh conditions on the leaves of field-grown plants, and survival may depend on tri-trophic interactions. Net growth of overtime -8. This overtime -12. The Salmonella belongs. In fact, numerous insects, such as flies, beetles and cockroaches, are associated with human habitations and livestock facilities, and have been linked with the spread of S. enterica [S. enterica and phytophagous insects [ Bacteria have evolved to exploit insects as hosts and/or vectors. Several studies have found an intimate relationship between insects and members of the Enterobacteriaceae , the famenterica . In all enterica -16. Seveenterica ,17. Inse insects .Escherichia coli. Wasala and collaborators [Musca domestica) represent a nutrient source that allows E. coli O157:H7 to multiply on spinach leaves. Additionally, Erickson et al. [E. coli O157:H7 populations on lettuce leaves that were inoculated soon after being fed upon by cabbage loopers (Trichoplusia ni). The effect of feeding by phytophagous insects on contaminated plants has not been studied, and the potential for insect activity to act as a ‘biomultiplier’ of S. enterica on agricultural crops remains unknown.  Specifically, insect-feeding activity may influence foodborne pathogen populations on leaves. Feeding sites could represent a preferential niche that would allow bacterial multiplication due to access to nutrients liberated from surrounding damaged plant cells or protein/carbon-rich substances excreted during or after feeding by insects ,19. The n et al.  observedS. enterica in the phyllosphere. We chose lettuce as our model phyllosphere because it is a common host to the three phytophagous insects we chose as representative cell-content and phloem-sap feeders [S. enterica with two types of insect feeding. Thrips are cell-content feeders that induce a condition described as ‘silvering’ on leaves, resulting from feeding damage using rasping-sucking mouthparts that damage surface epithelial cells. Hemipteran insects, such as aphids and leafhoppers, are phloem-sap feeders that ingest plant fluids without severe cellular damage to mesophyll cells. We found larger S. enterica populations on leaves co-infested with Macrosteles quadrilineatus. On plants infested with Frankliniella occidentalis, areas of silvering harbored higher S. enterica populations than areas without lesions. We also observed that insect feeding type did not influence insect contamination rates. However, S. enterica populations on individual insects varied by 2 logs.  In this study, we investigated the effect of feeding by phytophagous insects on survival of  feeders -22 and l feeders . BecauseS. enterica serovars Cubana strain 98A9878 [S. enterica strains were suspended from plates in sterile water to an optical density of 0.2 at 600 nm, which approximates 108 CFU/ml. S. enterica strains were always inoculated as a six-strain cocktail at 1:1:1:1:1:1 ratio per strain. A S. enterica strain cocktail was used to mitigate possible strain differences in the plant-microbe-insect interaction. Xylose Lysine Desoxycholate (XLD) agar (Difco), a Salmonella semi-selective growth medium in which all chosen strains produce black colonies, was used to determine S. enterica populations from both leaf and insect samples. To verify that the black colonies recovered with XLD were the inoculated strains, each strain was transformed with pKT-Kan that confers kanamycin resistance and constitutive green fluorescent protein expression [S. enterica on roots [Six  98A9878 , Enterit 98A9878 , Poona s 98A9878 , Baildon 98A9878  and Mbanpression  without on roots . Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) colony was maintained on green bean pods (Phaseolus vulgaris L.) on the campus of the University of Wisconsin, Madison, Wisconsin. F. occidentalis colonies were maintained in plastic deli cups under ambient temperature and a 16:8 (L:D) photoperiod as previously described [Macrosteles quadrilineatus Forbes (Hemiptera: Cicadellidae) was maintained on oat (Avena sativa L.) seedlings in a controlled environment with a 16:8 (L:D) photoperiod  [Myzus persicae Sulzer (Hemiptera: Aphididae) was kindly provided by Dawn M. Smith (Cornell University), and established and maintained on Chinese cabbage (Brassica rapa) under similar controlled conditions as the M. quadrilineatus colony on the campus of the University of Wisconsin, Madison, Wisconsin.A escribed . A colon°C dark) . A colonLactuca sativa cultivar butterhead), were cultivated in a growth chamber without insecticide treatments. Three-week-old plants were dip-inoculated with either sterile water, as a control, or a S. enterica cocktail suspension for 1 min. Plants were allowed to dry under a laminar flow hood and then kept in transparent plastic boxes at 25°C with covers on top to maintain high humidity for 24 h. S. enterica inoculum was verified by serial dilution and plated on LB-kan agar before and after plant dip-inoculation to ensure that the bacterial concentration was constant throughout the inoculation process. Lettuce plants , and periodically at 4, 9, and 13 dpi. At each sampling time, two 5 mm-diameter leaf discs were excised from each plant. All samples were homogenized in 500 μl of sterile water, and dilution plated on XLD-kan agar, and incubated at 37°C overnight for bacterial-population enumeration. From F. occidentalis-infested plants, leaf discs were sampled for S. enterica from leaf areas with visible F. occidentalis feeding damage (silvering). Each treatment consisted of 8 plants individually potted, which were randomly arranged and exposed to the same conditions. All experiments were repeated at least three times. Lettuce plants were dip-inoculated as described above. Twenty-four hours post inoculation, adult F. occidentalis were added to all plants. Leaf discs were sampled for S. enterica from areas with and without F. occidentalis feeding damage within the same leaf. Eight, individually potted plants were used for each experiment and the experiment was repeated three times.The same protocol was followed, as described above, in a separate set of replicated experiments (silvering +/-), except adult M. quadrilineatus and M. persicae were transferred to half of the Salmonella-inoculated plants and confined in individual clip-cages at a density of 4 M. quadrilineatus and 5 M. persicae per cage and 3 cages per plant. Empty cages were placed on insect-free plants. S. enterica populations were enumerated prior to insect introduction (0 dpi) and periodically at 2, 4 and 9 dpi as described above. Each treatment consisted of 8 plants planted in individual pots, which were randomly arranged and exposed to the same conditions. All experiments were repeated four times.In a separate set of replicated experiments, adult S. enterica inoculated plants were collected and examined microscopically with an Olympus BX-60 epifluorescence microscope . In order to identify preferred colonization sites on leaves from plants exposed and non-exposed to insects, leaf tissue was mounted on microscopic slides and examined for green fluorescence from bacteria as previously described [A subset of leaves from escribed .S. enterica-inoculated leaf at different densities due to different insect sizes . Petri dishes were sealed with a strip of parafilm to prevent insect escape. Live insects were collected in sterile microcentrifuge tubes after a 24 h exposure to S. enterica-inoculated leaves, placed at -80°C for 30 min to kill them without affecting potential surface contamination, and enriched in LB overnight at 37°C. Insects in enrichment broth were homogenized and a sterile loop was used to streak the enriched sample onto XLD-kan to verify the presence of S. enterica in or on insect bodies. Appearance of black colonies 24 h post-streaking were scored as S. enterica positive. A subset of random S. enterica presumptive positive, black colonies was confirmed by PCR using primers that target the invA gene of Salmonella as previously described [S. enterica and control leaves were not. Each treatment consisted of 10 petri dishes containing insects and the experiments were repeated four  or five (M. persicae and M. quadrilineatus) times, resulting in a minimum of 200 insects per treatment.Lettuce plants were dip-inoculated as described above. Ten leaves were carefully removed from control and inoculated plants 24 hours after inoculation with a sterile razor blade. Individual leaves were placed inside a sterile petri dish. Non-contaminated insects were collected from respective colonies, and placed in the bottom of each dish containing either the mock or a escribed . AdditioS. enterica population size per insect was determined following feeding on contaminated produce. Specifically, green bean pods were surface-sterilized by dipping in 10% bleach solution for 10 min, and placed in individual 50 ml conical tubes containing either 6 ml of sterile water or S. enterica suspension (prepared as described above). Conical tubes were placed horizontal in a shaking incubator at 37°C at 200 rpm overnight. Green beans were removed from the liquid and allowed to dry under a laminar flow hood and then placed in new sterile conical tubes. F. occidentalis were collected from the corresponding colony, and added to the conical tubes containing either the mock or the S. enterica-inoculated beans at a density of 15 F. occidentalis per tube. F. occidentalis-proof mesh was fixed to the tube cap to prevent insect escape or death from suffocation. Furthermore, green beans samples were collected, serially diluted, and plated on XLD-kan before insects were added and after they were collected, to verify that inoculated green beans were contaminated with S. enterica and control green beans were not. Live insects were collected in sterile microcentrifuge tubes after a 24 h contamination period, and placed at -80°C for 30 min to kill them without affecting potential surface contamination. Then, insects were homogenized in sterile water and S. enterica populations per insect were enumerated directly on XLD-kan. A subset of random S. enterica presumptive positive, black colonies was confirmed by PCR as described above. Each treatment consisted of 6 conical tubes and the experiment was repeated three times, resulting in a minimum of 100 insects per treatment.S. enterica differed between treatments or over time, analysis of covariance (ANCOVA) was used to test the potential effects of insect feeding on S. enterica populations on leaves, with treatment and time (dpi) as covariates. Bacterial counts were log transformed prior to analysis and repetitions of the experiment were considered as block factors. In this manuscript, the intercept parameter is described as the starting S. enterica population, and the slope parameter is described as a measure of S. enterica population persistence. In the special instance where both silvered and non-silvered leaf tissue was sampled, leaf samples from the same plants were randomly assigned to one of the two treatments prior to insect addition. Therefore, in the analysis of the silvering assay, the intercept was estimated in the same way, but the model was modified to disallow variation between treatments for the y-intercept. For the insect contamination experiments, a two-tailed Z test  was used to test if the percentage of contaminated insects was statistically higher than 50% of the total population of insects tested. All statistical analysis were performed using R software [To determine whether the average population or incidence of software .S. enterica were exposed to F. occidentalis to investigate if insect infestation influenced S. enterica populations. Although plants were inoculated at a high concentration (108 CFU/ml), bacterial populations that colonized leaves at the beginning of the insect infestation interval (0 dpi), averaged 104 CFU/mm2. S. enterica was not recovered from uninoculated control plants (data not shown). In the case of inoculated plants, S. enterica populations declined over time. Surprisingly, the final bacterial concentrations did not reach zero, even 14 days after inoculation. In addition to differences in bacterial populations over the sampling interval (dpi P<0.05); the slopes varied among experimental replications  or population decline over time among plants exposed or non-exposed to F. occidentalis, evident in the interaction trt:dpi  on the same leaf potentially increasing the variability among samples. Lettuce plants inoculated with  P>0.05, . F. occigfp-tagged S. enterica cells accumulating in areas that were fed upon by F. occidentalis (data not shown); therefore, the effect of F. occidentalis feeding damage was further investigated. Survival of S. enterica in feeding areas with obvious silvering was compared with areas without visible feeding damage in the same plant. Similar to our earlier findings, bacterial populations declined over the sampling interval (dpi P<0.05). But in contrast to the experiments with or without F. occidentalis, bacterial populations were similar among experimental replications (P>0.05) in these experiments. Overall, S. enterica population decline was delayed in areas with silvering , and sustained significantly higher bacterial populations even at 10 and 13 dpi when compared to undamaged areas  or in population decline over time among plants exposed or non-exposed to M. persicae . In contrast, exposure of plants to M. quadrilineatus enhanced S. enterica survival compared to plants that were not exposed to insects , resulting in approximately half a log higher bacterial populations at 13 dpi in M. quadrilineatus-exposed plants. To determine if  P<0.05; . LikewisS. enterica from contaminated plant material was observed by detection of the bacteria in approximately 50% of all insects tested. A total of 241 F. occidentalis, 229 M. quadrilineatus, and 289 M. persicae were exposed to contaminated lettuce leaves for 24 h, and subsequently tested for the presence of S. enterica. Dead insects were not collected to assure that sampled insects were in contact with contaminated plant tissue. S. enterica was not isolated from untreated control treatments. From the exposed insects, 52% of F. occidentalis and M. persicae were positive for S. enterica, while in the case of M. quadrilineatus, the contamination rate was slightly lower (47%). Hypothesis testing with Z-scores  suggest that rates of contamination were not significantly different when compared to the remaining 50% of the corresponding insect population. The potential for phytophagous insects to become contaminated with S. enterica, S. enterica populations were enumerated from individual insects following feeding on contaminated plant tissue. F. occidentalis were used in this experiment because they are relatively tiny and slender , and represent the smallest in size of the insects evaluated in this study. S. enterica was not isolated from untreated control treatments. Based on direct plating, 68% (98 out of 145) of the F. occidentalis tested positive for contamination of S. enterica, while 32% (47 thrips) tested negative  of the F. occidentalis harbored more than 102S. enterica CFU on their bodies , as cell rupture feeders, not salivary sheath feeders [M. quadrilineatus used an intermediate feeding strategy that allowed the enhanced persistence of S. enterica by causing less drastic mesophyll cell damage and release of phloem-sap contents. Additionally, it is possible that the larger stylets of M. quadrilineatus, compared to M. persicae, caused more physical damage to plant tissues and introduced more S. enterica cells into damaged tissues, where the pathogen had access to nutrients. Feeding behavior, instead of mouthpart type, may correlate with human bacterial pathogen survival in infested leaves. Miles  describe feeders ,44. This feeders . AlthougM. quadrilineatus feeding could have indirectly benefited S. enterica colonization of lettuce plants by antagonizing defense responses associated with pathogen establishment and infection, such as SA-dependent and –independent defenses and pathogenesis related proteins. Plant response to thrips and aphid feeding involves several signaling pathways associated with both pathogen infection and wounding [Rosa hybrida cv. Sonia) plants previously infected with a plant pathogen could have had an adverse effect on aphids and thrips feeding. However, Erickson and collaborators observed lower E. coli O157:H7 populations internalized within leaves previously exposed to insects, including aphids and thrips [S. enterica populations, among plants exposed to F. occidentalis or M. persicae, were similar to those without insects, suggesting that defense responses induced by aphids or thrips do not have a relevant effect on S. enterica populations. Moreover, whether S. enterica contamination of plants positively or negatively affects the behavior of phytophagous insects remains unknown. Plant defense response to herbivores may influence human pathogen populations. Feeding strategies that cause more aggressive damage to plant tissue, such as chewing, rasping-sucking, or repeated perforation of multiple plant cells stimulate the plant’s jasmonic acid dependent and -independent wound-responses ,45. Sevewounding . Moreovewounding  reportedd thrips . In our S. enterica on lettuce. However, the ability of phytophagous insects to become contaminated with the human pathogen seems to be independent of feeding strategy used. It was demonstrated that F. occidentalis, M. quadrilineatus, and M. persicae could become contaminated with S. enterica from contaminated plant tissues including lettuce leaf and green bean pods. Particularly, F. occidentalis, the smallest of the insects tested, harbored large S. enterica populations after a 24 h access period to contaminated plant material. It is well know that adult thrips are not strong flyers; however, they are quite active and can move quickly on the surface of leaves. In fact, the wandering behavior, which involves roaming, scraping of their heads, and search for new-feeding sites, is characteristic of thrips when they are not probing [S. enterica-adhered to their bodies over leaf surfaces and flowers. It is acknowledged that the use of high inoculum concentrations that are unlikely to occur in natural environments could have increased the probability of movement of bacteria by insects. Nevertheless, it is important to emphasize that bacterial populations at the time of infestations were similar to concentrations of Salmonella recovered from drainage water [S. enterica. Although in this study it was not determined whether S. enterica could adhere to the outside of phytophagous insects or be ingested, the potential for these insects to be biological vectors of S. enterica remains to be determined. Furthermore, since S. enterica was recovered from insect bodies and insect damaged plant material, insects or insect-damaged plant tissue could be exploited as a novel sentinel strategy for S. enterica-contaminated crop monitoring. It seems likely that only insect feeding behaviors that cause direct damage to plant cells tended to enhance the longevity of  probing . This, i probing , suggestge water  and wounge water . Taken t


Vibrio cholerae, is responsible for several million cases and over 120,000 deaths annually. When contaminated water is ingested, V. cholerae passes through the gastric acid barrier, penetrates the mucin layer of the small intestine, and adheres to the underlying epithelial lining. V. cholerae multiplies rapidly, secretes cholera toxin, and exits the human host in vast numbers during diarrheal purges. How V. cholerae rapidly reaches such high numbers during each purge is not clearly understood. We propose that V. cholerae employs its bactericidal type VI secretion system to engage in intraspecies and intraguild predation for nutrient acquisition to support rapid growth and multiplication.Diarrheal diseases are the second-most common cause of death among children under the age of five worldwide. Cholera alone, caused by the marine bacterium  Vibrio cholerae – the causative agent of the diarrheal disease cholera – is an ancient companion of human civilization. Reports of cholera symptoms date back to ancient Greek civilization and Sanskrit writings , a horizontally acquired DNA element, contains several genes for sialic acid transport and catabolism  that also functions as the adhesin for attachment to mucin in the host intestines  screen employing non-toxigenic V. cholerae strains carried out in human volunteers suggested that T6SS genes are transcribed in the human host (V. cholerae strain that does not express the secretion apparatus under laboratory conditions (V. cholerae T6SS expression in vivo.To engage in predation , V. chol complex . This hi complex . The disscoideum . Recentlscoideum , pore-foscoideum , and pepscoideum  were fount mouse  and infant mouse  cholera man host . These fnditions . CollectV. cholerae in the marine environment induce T6SS biosynthesis (V. cholerae strains that otherwise do not express the secretion apparatus under laboratory conditions. In addition, we recently showed that non-toxigenic V. cholerae strains isolated from the Rio Grande Delta express their T6SS constitutively (V. cholerae’s T6SS immunity system (V. cholerae and that T6SS-mediated selective intraspecies killing permits strains to distinguish self from non-self (Evidence that T6SS genes are also likely expressed in the environment emerged from a report showing that salt concentrations and temperatures analogous to those encountered by ynthesis . These etutively  supportiy system  mediatesnon-self .Vibrio cholerae does not utilize the T6SS exclusively to kill bacteria of its own species; several additional prokaryotes are susceptible to T6SS-dependent killing. Some of these susceptible bacteria include V. communis, V. harveyi, V. mimicus, V. fischeri,and Pseudoalteromonas phenolica isolated from the same environmental niches as V. cholerae (V. cholerae in the marine environment; these include Escherichia coli, Salmonella typhimurium, and Citrobacterrodentium. Thus far, only Gram-negative bacteria have demonstrated susceptibility to V. cholerae T6SS killing, while Pseudomonas aeruginosa was recently shown to be capable of counter attacking cholera bacteria utilizing its own T6SS (Enterococcus faecalis, Bacillus subtilis, Listeria monocytogenes,and Staphylococcus aureus are resistant to V. cholerae T6SS bactericidal activity (V. cholerae’s T6SS’s bactericidal effect; alternatively we cannot rule out the possibility that additional effectors targeting Gram-positive species remain to be discovered and characterized. Either way, susceptibility of the host microbiome to T6SS-mediated killing may be one of numerous factors contributing to V. cholerae’s ability to successfully colonize the host.cholerae . Other bown T6SS . Gram-poactivity . One posBacteroides, Firmicutes, and Actinobacteria phyla are displaced while harmful Proteobacteria prosper (Escherichia coli K12 capable of colonizing the human small intestine implanted at high inocula failed to maintain a foothold in the intestines of volunteers suffering from acute cholera (V. cholerae T6SS killing (V. cholerae engage their T6SS in intraspecies competition by killing cholera strains harboring different effector/immunity proteins that compete for the same niche in the small intestine upon entry into the host, leading to kin selection and clonal expansion. Subsequently, killing of T6SS-sensitive bacteria competing for the same niche in the large intestine may allow V. cholerae to engage in intraguild predation thereby shifting bacterial populations to its advantage and acquire nutrients from prey. For either scenario, we suggest that V. cholerae’s T6SS-mediated bacterial killing has contributed to its success as an enteric pathogen.The host microbiome experiences profound changes during cholera infections. Commensal bacteria belonging to the  prosper . Benign  cholera . This ou killing . While sThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Daniele Provenzano and Stefan Pukatzki wrote the manuscript and conceived the ideas therein.


Rickettsia felis in the Canary Islands. Antibodies against R. felis were found in 5 adsorbed serum samples from 44 patients with clinically suspected rickettsiosis by Western blot serology. Fleas from 1 patient's dog were positive for R. felis by polymerase chain reaction.We report the first cases of human infection by  Rickettsia felis is an intracellular bacterium   as the main vector  , autochthonous cases of murine typhus have been reported  in the Canary Islands. Among the 44 patients, 24 showed a positive serologic result. Antibodies against other agents  were also tested; all were negative.Forty-four patients were recruited for a prospective study of fever of intermediate duration . Demographic, clinical, and laboratory data were collected for all patients. Chest radiographs and blood and urine cultures were taken. Antibodies against R. felis, all serologic results were confirmed by microimmunofluorescence (MIF) in France, as previously described  (3). DNA was extracted and amplified with primers that targeted the citrate synthase sequence, as previously described (http://www.ncbi.nlm.nih.gov/BLAST/).Five fleas from the dog of 1 R. felis infection, 13 with the diagnosis of R. typhi infection, and 13 with the diagnosis of indeterminate rickettsioses  to R. conorii and R. typhi (2 of 5 patients) than did patients with R. typhi infection (0 of 13). On the basis of R. felis data, we conclude that patients with R. felis infection may have no cross-reactivity with other rickettsiae, cross-reactivity with SFG rickettsiae, or cross-reactivity with both SFG rickettsiae and R. typhi. Genetic support for cross-reactivity with R. conorii is plausible because most membrane proteins of SFG and R. felis are extremely close (surface cell antigen [Sca] family). Genome analysis showed that several genes were present in R. felis and R. typhi and absent for other SFG, which could explain the cross-reactivity between R. felis and R. typhi (R. felis and other SFG and typhus group rickettsiae are observed, only Western blot and cross-adsorptions will allow a specific diagnosis once reactivity has disappeared after adsorption with R. felis antigen. By contrast, a band of ≈31 kDa for the R. felis antigen persists after adsorption with R. conorii and R. typhi.Cross-reactions in serologic testing for