Pieris rapae, for plants occupied by combinations of conspecific larvae, heterospecific larvae (Plutella xylostella), specialist parasitoids of Pi. rapae (Cotesia glomerata) and generalist predators (ants). We previously reported that the females showed equal preference for Pl. xylostella-infested and uninfested plants. Here, we showed that Pi. rapae females preferred uninfested plants to conspecific-infested ones, and Pl. xylostella-infested plants to Pi. rapae-infested ones. We discuss these oviposition preferences of Pi. rapae females in the framework of costs and benefits of interspecific herbivore associations from the above point of view.When deciding where to oviposit, herbivorous insects consider: (i) the plant’s value as a food source, (ii) the risks of competing with con- and heterospecific herbivores, and (iii) the risks of parasitism and predation on the host plant. The presence of con- and/or heterospecific competitors would further affect the oviposition preference, because the preceding herbivores induce direct/indirect defences in plants against forthcoming herbivores, and thereby alter oviposition decisions. In previous studies, the abovementioned factors have not been studied in an integrative manner. We performed here a case study of this by assessing the oviposition preferences of a small white butterfly,  Because caterpillars have low mobility, their mother influences their developmental success by her choice of plants on which to oviposit. We reported that Pl. xylostella females preferentially oviposited on plants infested by Pi. rapae larvae [Cotesia vestalis (Hymenoptera: Braconidae), a dominant parasitoid of Pl. xylostella larvae, is lower on plants infested by the two species of caterpillars together than on plants infested by Pl. xylostella larvae alone because of the reduced attraction of C. vestalis to plants infested by both species [Pl. xylostella larvae was mediated by the previous infestation by Pi. rapae larvae. This case study shows that enemy-rare space caused by heterospecific-herbivore-induced indirect defence affects oviposition preference of other species of herbivores (herbivore-induced enemy-rare space).For herbivore-induced indirect defence by attracting natural enemies, most previous studies have focused on a defence against a single herbivore species –16, and e larvae . This pr species . Thus, tPi. rapae show equal oviposition preference between Pl. xylostella larvae-infested cabbage plants and uninfested cabbage plants, and that resource competition and preference-performance do not explain this equal preference [Pieris rapae females should oviposit eggs on uninfested plants, because cabbage plants infested by either Pi. rapae larvae, Pl. xylostella larvae or both attract Cotesia glomerata at the same intensity and at the same levels of damage; thereby, Pl. xylostella larvae-infested plants are herbivore-induced enemy-dense space. One of the objectives of this study is to try to explain this paradox. To do so, in this study, we focused on apparent interactions .Pi. rapae females in habitats in which conspecific herbivores, heterospecific herbivores (Pl. xylostella larvae), specialist parasitoids (C. glomerata) and generalist predators  are involved. By integrating potential factors affecting oviposition preference by herbivores, we show that Pi. rapae is tuned to a particular structure of tritrophic interaction webs in which interspecific herbivore associations are active.Here, we investigated factors affecting the oviposition preference of 2.2.1Brassica oleracea cv. Sikidori, that were more than one month old (with ca five leaves) were grown in a climate chamber at 25±2°C, 60% relative humidity, and 16 L:8 D fluorescent light (5500 lux). We collected adult females of Pi. rapae from a field near Kyoto City, Japan, and allowed them to lay eggs on these cabbage plants in the laboratory. Hatched Pi. rapae larvae were reared on detached cabbage leaves in the climate-controlled room . Plutella xylostella larvae were collected from cruciferous plants in a field near Kyoto City and reared on cabbage leaves in the climate-controlled room under conditions identical to those for Pi. rapae larvae.Cabbage plants, Pi. rapae, we collected adult females in the field and held them for 1 day in a cage without crucifer plants. Because Pi. rapae females mate soon after emergence, it was reasonable to assume that the collected females had already mated .To test the oviposition preference of Cotesia glomerata were obtained from parasitized Pi. rapae larvae collected in a field near Kyoto, Japan. Adult wasps of both sexes were kept in a climate-controlled room , where they were contained in a plastic cage (13×20×13 cm) with a droplet of honey as an energy source for a period of 3 days. After mating, females were transferred to a glass tube with a small amount of honey and kept in a climate-controlled room at 18±2°C, 50–70% relative humidity, and continuous darkness. They were used for experiments within 10 days of maturation. At least 1 h before the start of each experiment, oviposition-inexperienced females were transferred to another climate-controlled room  under 16 L:8 D light conditions.Lasius japonicus, Pristomyrmex pungens and Paratrechina flavipes; identified by Dr Y. Takematsu, Yamaguchi University, Yamaguchi, Japan) were sampled from a field on the campus of Kyoto University and allowed to establish new colonies in a plastic cage (10×20×3 cm) in the laboratory . The ants were fed a sucrose solution and mealworms in a climate-controlled room  until use in experiments one week after founding the new colony.Colonies of three ant species (Hymenoptera: Formicidae: 2.2Pi. rapae in a cage (180×180×180 cm) covered with gauze (mesh size: 1.8 mm) was assessed. The cage was positioned in an experimental field on the campus of Kyoto University. Ten mated females of Pi. rapae were released into the cage from 09.30 to 15.30. They were allowed to oviposit on two plants that received contrasting treatments according to one of the following two experimental designs: (i) an uninfested plant versus a plant infested by conspecific larvae, and (ii) a plant infested by conspecific larvae versus a plant infested by larvae of Pl. xylostella.Oviposition preference of Pi. rapae larvae- or Pl. xylostella larvae-infested cabbage plants at the early stage were 10–30% of the leaf surface. Based on this, we used intermediate damage levels (15–20%) to test the oviposition preference. Larvae-infested plants were prepared by releasing either five second-instar Pi. rapae or 20 second- or third-instar Pl. xylostella randomly over the leaves of a plant, where they were kept for 24 h, resulting in consumption of 15–20% of the leaf surface. For both types of infested plants, all herbivores were removed at the end of the 24 h feeding period.Visual observations in our study field showed that the naturally occurring damage levels of Pi. rapae are usually laid singly on plants [G-test [The experiments were performed on sunny days 20–30°C) between June and October, and each choice test was replicated three times. As eggs of –30°C betn plants . For stan plants ,20, we e [G-test .2.3Pi. rapae in their larval stage. To do this, we used a first-instar Pi. rapae that was inoculated on a plant that had been infested 1 day earlier by one other first-instar Pi. rapae, or on plants that had hosted no conspecific larva. After the inoculation, the initially inoculated occupant was not removed. The initial and the later occupant were discriminable owing to their size difference and the duration of the larval period. The later occupant was reared until pupation. At this point, more than 60% of the leaf surface of a plant was consumed. The experiment was replicated on 20 plants for each treatment in a climate-controlled room . We compared pupal weight and duration of the larval period between uninfested plants and plants infested by a conspecific larva using a Student’s t-test for comparison of means. We also compared the survival rates by Fisher’s exact test.We tested the effects of the presence of a conspecific larva on the same plant on the performance of 2.4Pi. rapae larvae- or Pl. xylostella larvae-infested cabbage plants of early stage were 10–30% of the leaf surface under field conditions. Thus, we compared 10%- and 30%-damaged plants. Larvae-infested cabbage plants were prepared as follows. Three second- or third-instar Pi. rapae were released randomly on the plant and allowed to feed there for 24 h. After 24 h, ca 10% of the leaf surface of the plant had been consumed. We then removed the larvae and their associated products  from the leaf with a small, fine brush. To obtain cabbage plants with 30% damage, we released 10 instead of three second- or third-instar Pi. rapae.As mentioned above, the naturally occurring damage levels of C. glomerata to Pi. rapae-infested plants with 10% and 30% damage were observed in a cage (25×35×30 cm) with three windows covered by nylon gauze provided with a single door. The cages were placed together in a climate-controlled room . A detailed description of the experimental procedure is given by Shiojiri et al. [G-test [The flight responses of females of i et al. . Each wa [G-test . Wasps t2.5L. japonicus, Pr. pungens and Pa. flavipes for one larva of Pi. rapae versus one of Pl. xylostella were assessed in two-choice experiments carried out in the feeder set-up . An aqueous solution of sugar and water in Petri dishes (4 cm in diameter) was supplied. The dish was situated in a climate-controlled room . We also scored which of the two larvae was contacted first and predated by an individual ant first. We used second-instar Pi. rapae larvae and third-instar Pl. xylostella larvae because they were quite similar (5–6 mm) in length. Predation (or contact) scores were then tested against the null hypothesis that the predation events were binomially distributed with equal probability for each of the two species of larvae.Predation preferences of r set-up a,b. An aPr. pungens on Pi. rapae larvae when offered together with various numbers of Pl. xylostella larvae was also examined in the feeder set-up. Five second-instar Pi. rapae and 0, 5, 10 or 15 third-instar Pl. xylostella were placed in the feeder. Every 15 min, we recorded the number of Pi. rapae larvae present in the feeder. Using the same procedure, we also carried out the test with 15 third-instar Pl. xylostella and various numbers  of Pi. rapae larvae. We repeated each experiment five times, and subjected the data of each observation record to Tukey’s honest significance test (HSD) (α=0.05).The predation rate of Pi. rapae larvae and Pl. xylostella larvae was also tested in a cage (180×180×180 cm) in the experimental field of Graduate School of Agriculture, Kyoto University. Inside the cage, there were ant nests . We placed a glass Petri dish  containing a cabbage leaf (ca 6 cm long), 10 second-instar Pi. rapae and 10 third- to fourth-instar Pl. xylostella in the experimental field cage covered with gauze (mesh size=1.8 mm). No crucifer plant was grown in the cage. Then, 1.5 and 3 h after the start of the experiment, we placed a small amount of sugar crystals (ca 3 g) in the Petri dish, observed the number of surviving larvae of each herbivore species and prevented any other arthropod predator from entering the cage. Each experiment was repeated four times. For each observation time, the numbers of surviving larvae of each herbivore species were subjected to the Wilcoxon–Mann–Whitney test on the differences in survival rates between the two species.Predation by ants on 3.3.1Pi. rapae females showed different oviposition preferences for the same plant species with different infestation histories. Pieris rapae females significantly preferred uninfested plants to Pi. rapae-infested ones . The significance of GH was due to the fact that the first experiment was not significant , whereas other two experiments were highly significant . Comparison of the attraction of Pi. rapae with Pl. xylostella-infested plants and Pi. rapae-infested plants revealed that Pi. rapae preferred the former .In this study, we asked whether 3.2Pi. rapae larvae among uninfested, Pl. xylostella-infested, and Pi. rapae-infested plants. Previously, we reported that the performance of Pi. rapae larvae on Pl. xylostella-infested plants and on uninfested plants were not significantly different [Pi. rapae larvae on Pi. rapae-infested plants and on uninfested plants and found that the number of larvae surviving until pupation on uninfested plants was 16 (n=20) and on Pi. rapae-infested plants was 18 (n=20) and thus not significantly different . Furthermore, no significant difference was observed in pupal weight  or duration of the larval stage  between Pi. rapae reared on uninfested cabbage plants and on cabbage plants infested by Pi. rapae larvae  over those with 10% infestation . The results suggested that the offspring of a Pi. rapae female on a plant already infested by Pi. rapae larvae would suffer higher parasitism by C. glomerata than Pi. rapae larvae on an uninfested plant that had not suffered caterpillar damage by previous infestation of Pi. rapae larvae.We tested whether specialist parasitoids affect the oviposition preference of 3.4Pi. rapae. Here, we focused on ant species that are major predators of the larvae under field conditions. All three ant species  significantly preferred Pl. xylostella larvae to Pi. rapae larvae in the two-choice tests  and the number of Pl. xylostella larvae was varied from 0 to 15, predation on Pi. rapae larvae significantly decreased with increasing numbers of the latter . When the number of Pl. xylostella larvae was constant (i.e. 15) and the number of Pi. rapae larvae was varied , there were no significant differences in the predation rate on the former with increasing numbers of the latter .Next, at different constant supplies of Pi. rapae and Pl. xylostella larvae were also assessed in the field. The ant species observed in the study field were Pa. flavipes, L. japonicus and Formica japonica. At 1.5 h after the start of the experiment, no significant difference was observed in the number of surviving larvae between Pi. rapae and Pl. xylostella . However, after 3 h only 42.5% of Pl. xylostella larvae survived, whereas 80% of Pi. rapae larvae survived  of d plants . Thus, i4.1Pieris rapae females preferred uninfested cabbage plants over those infested by conspecific larvae (a). Sato et al. [Pi. rapae females for uninfested wild Rorippa indica (Brassicaceae). Intraspecific resource competition may explain the preference for uninfested plants. Another possible explanation is that avoiding plants with conspecific larvae prevents the eggs being cannibalized by conspecific larvae (intraspecific predation) [c larvae a. Sato eo et al.  observededation) .Pi. rapae females for uninfested plants. The parasitism rate of Pi. rapae larvae by C. glomerata can sometimes be considerable; it varies in time and may occasionally exceed 70% [C. glomerata preferred plants infested by Pi. rapae larvae, its host, to uninfested plants [C. glomerata with increasing damage by host larvae. Thus, when eggs are deposited on cabbage plants infested by conspecifics, the emerging larvae may face an occasionally higher risk of C. glomerata parasitism than those hatched from eggs deposited on uninfested plants.In addition to these direct effects, indirect effects may also explain the preference of ceed 70% . We prevd plants , and we 4.2Pi. rapae females preferred Pl. xylostella-infested to Pi. rapae-infested plants (b). In this preference, what matters to Pi. rapae females is not how much competition their larvae will experience immediately on the plant but rather how much they will suffer from predation before reaching maturity. Interspecific differences in the rate at which plant damage accumulates during larval development are important and should be taken into account. In this respect, the two herbivore species were extremely dissimilar; the total amount of damage done by one Pi. rapae larva before pupation to yellow cress plants (R. indica) [ca 25 times higher than that by one Pl. xylostella larva to cabbage seedlings [Pi. rapae-infested plants would incur more total damage than Pl. xylostella-infested ones (intraspecific resource competition). The assumption that Pi. rapae females do not select oviposition sites simply based on the leaf area presently available as a resource is also supported by the fact that these females preferred artificially damaged wild R. indica to those that received the same amount of damage caused by feeding of conspecific larvae [We found that d plants b. In thi indica)  is ca 25eedlings . Thus, Pc larvae .C. glomerata, irrespective of the parasitoid’s target herbivore species .The amount of damage to a plant is positively correlated with its attractiveness to the parasitoid tudy and ,27). ThuPi. rapae females preferred Pl. xylostella larvae-infested plants to Pi. rapae larvae-infested plants, we studied the roles of generalist predators (ants) shared by the two herbivore species. Direct observation showed that ants of three species preferred to prey on Pl. xylostella larvae first . This reduced predation by ants on Pi. rapae larvae could not simply be based on an increase in the total number of larvae, because adding more Pi. rapae larvae did not affect the risk of predation on Pl. xylostella(b). We therefore hypothesized that ants preferred Pl. xylostella larvae to Pi. rapae larvae. In the field experiments , ants prefer Pl. xylostella larvae when both herbivore species are present on the same plant . In the laboratory experiment, all larvae were consumed by the ants within 2 h  the distance between prey and any ant nest was larger in the field than in the laboratory. Therefore, the predation pressure by ants on Pi. rapae larvae would be substantially reduced when they occur together with Pl. xylostella larvae on the same plants under natural conditions (apparent competition). All these factors would explain why females of Pi. rapae deposited fewer eggs on conspecific-infested plants than on Pl. xylostella-infested plants.To further determine why ae first . Furthera larvae a. This rylostellab. We theeriments , ants fee larvae , which ae larvae ,30. Becathin 2 h , whereasll alive . This pr4.3Pi. rapae females showed no preference when offered Pl. xylostella-infested plants versus uninfested plants [Pi. rapae and Pl. xylostella larvae compete for the same resources. Intraguild predation was unlikely, because Pl. xylostella larvae do not prey on Pi. rapae eggs . For indirect effects, Pl. xylostella-infested plants attracted C. glomerata to the same degree as Pi. rapae-infested plants [Pl. xylostella-infested plants would result in a higher incidence of parasitism (herbivore-induced enemy-dense space). All these data suggested that Pi. rapae females preferred uninfested plants to those infested by Pl. xylostella, but the observed results were opposite [Pl. xylostella, because of the positive effect of apparent competition through generalist predators (i.e. ants) that might compensate for the resource competition and apparent competition mediated by specialist predators, such as C. glomerata.We previously reported that d plants . Clearlyd plants ; therefoopposite . Here, w4.4Pi. rapae females always disperse from their natural habitat, even if that habitat still contains enough food for their offspring [Pi. rapae females finding habitats in which C. glomerata, a specialist parasitoid of their larvae, is absent or rare [Pl. xylostella larvae) and other—usually generalist—predators (e.g. ants) may prevail. Consequently, the first colonizers of this new habitat need to cope only with the risk of being eaten by generalist predators, and to compete with other herbivores on the same plants. By contrast, females of Pi. rapae that arrive later are also threatened by the specialist parasitoid C. glomerata that will follow the invaders, albeit with a delay. Furthermore, females of Pl. xylostella preferred to lay eggs (future competitors) on plants already infested with Pi. rapae larvae [Newly emerged ffspring ,31. This or rare . In suche larvae .Pl. xylostella and Pi. rapae larvae probably co-occur on specific plants, and in this situation, both are threatened by their respective specialist parasitoids and generalist predators, ants. When co-occurring, the presence of Pl. xylostella larvae increases the incidence of parasitism of Pi. rapae larvae by C. glomerata, because the plant volatiles become more attractive to the parasitoid, but the rate of predation by ants declines (a). By contrast, the coexistence of Pi. rapae and Pl. xylostella larvae on a plant results in a decreased incidence of parasitism of Pl. xylostella by Cotesia plutellae, a specialist parasitic wasp of Pl. xylostella larvae [Pl. xylostella larvae would not be changed (b).Under these conditions, declines a. By cona larvae , but eff changed b.Pi. rapae, for plants occupied by combinations of conspecific larvae, heterospecific larvae (Pl. xylostella), specialist parasitoid C. glomerata and generalist predators (ants). As is illustrated by our case study, the value of a host plant to a female herbivore depends on trophic interactions , as well as on non-trophic interactions (inter- and intraspecific resource competition and intraguild predation). Recently, it has been shown that egg deposition stimuli change the physiology of plants and subsequently the performance of hatched larvae and their natural enemies [We studied here the oviposition preferences of a small white butterfly,  enemies –35. Such


Porphyromonas gingivalis, Tannerella forsythia and Filifactor alocis, as potential pathogens that were significantly associated with periodontitis and with two periodontitis diagnostic parameters (pocket depth and attachment loss) in both saliva and supragingival plaque habitats. Interestingly, pairwise correlation analysis among the 21 OTUs revealed that Filifactor alocis was positively correlated with seven other putative pathogens , forming a co-occurrence group that was remarkably enriched in all three habitats of periodontitis patients. This bacterial cluster showed a higher diagnostic value for periodontitis than did any individual potential pathogens, especially in saliva. Thus, our study identified a potential synergistic ecological pattern involving eight co-infecting pathogens across various oral habitats, providing a new framework for understanding the etiology of periodontitis and developing new diagnoses and therapies.Periodontitis is a highly prevalent polymicrobial disease worldwide, yet the synergistic pattern of the multiple oral pathogens involved is still poorly characterized. Here, saliva, supragingival and subgingival plaque samples from periodontitis patients and periodontally healthy volunteers were collected and profiled with 16S rRNA gene pyrosequencing. Different oral habitats harbored significantly different microbiota, and segregation of microbiota composition between periodontitis and health was observed as well. Two-step redundancy analysis identified twenty-one OTUs, including  Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. More recently, the advent of high-throughput next generation sequencing techniques allowed us to characterize the overall structure of the complex oral microbial ecosystem at the OTU level, confirming the association of those potential pathogens with periodontitis and revealing additional periodontitis-associated phylotypes such as Prevotella spp., Porphyromonas endodontalis, Filifactor alocis, and Peptostreptococcus stomatis1011121314Periodontitis is a polymicrobial disease of the oral cavity and a risk factor for chronic diseases such as cardiovascular disease and diabetes mellitus124678The oral cavity is a dynamic ‘continuum with the external environment'1718et al. defined subgingival bacterial communities by checkerboard DNA-DNA hybridization and observed ecological interbacterial relationships among 32 examined species. They found that those species could be clustered into five complexes in which members showed highly similar distribution in subgingival microbiotaPolymicrobial diseases involve co-infection with multiple bacteria, which may act synergistically to contribute to disease progression and clinical outcomes56in situ. Instead, recent studies have mainly focused on interactions between putative pathogens using in vitro or animal models. For instance, a rat model of periodontitis was used to study the synergistic virulence of P. gingivalis, T. denticola and T. forsythia, demonstrating that those three pathogens existed as a consortium and synergistically increased alveolar bone resorption in co-infected ratsin vitro flow cell biofilm model demonstrated that P. gingivalis and T. denticola exhibit a strong synergy in polymicrobial biofilm formationP. gingivalis can interact synergistically with other pathogens, but not whether this pathogen actually co-exists and co-infects with the other tested pathogens in situ. Many putative pathogens have been associated with periodontitis. Whether these bacteria contribute to periodontal disease in random combinations or via specific synergistic patterns still remains a fundamental question.After that initial effort to elucidate the ecological relationships of oral microorganisms, few studies explored the inherent coexistence and distribution patterns of multiple pathogens in human oral samples Filifactor alocis-centered co-occurrence group of potential pathogens that was significantly enriched in periodontitis samples. These results laid a microbial ecological foundation for future etiological studies in periodontitis.Using 16S rRNA pyrosequencing, we obtained a comprehensive view of the oral bacterial communities in saliva, supragingival plaque and subgingival plaque from Chinese adults with and without periodontitis. We identified periodontitis-associated bacterial species that are commonly shared across different oral habitats, and we detected a A total of 307,679 usable raw pyrosequencing reads passing the quality control standards were obtained from 150 samples, with an average of 2051 reads per sample see  online. As revealed by the Shannon diversity index, supragingival plaque possessed the highest level of bacterial diversity, whereas saliva showed the lowest diversity. A higher level of diversity was observed in periodontitis individuals than healthy controls in both saliva and supragingival plaque . The SimPrincipal coordinate analysis (PCoA) based on weighted UniFrac matrices revealed remarkable differentiation of the bacterial communities in the five groups, and the differences between oral habitats were more distinct than those between patients and healthy controls of the same habitats . These fBacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria and Spirochaetes were the most predominant phyla when the three oral habitats were considered together, contributing to 39.1%, 23.2%, 13.4%, 11.4%, 8.3% and 2.1% of all reads, respectively.Thirteen phyla with 143 genera were detected in the oral microbiota. Streptococcus, Leptotrichia and Actinomyces accounting for 18.90% of all reads-were stable in subjects with different periodontal health status, as revealed by the similar relative abundances in saliva and supragingival plaque from periodontitis and healthy cohorts. These three genera represent the core members of the oral microbiota. The remaining six genera, including Prevotella, Porphyromonas, Capnocytophaga, Fusobacterium, TM7_genera_incertae_sedis and Clostridium XIVa displayed significantly different abundance  between health and periodontitis , constituting the predominant oral inhabitants. Among them, three genera-itis see  online.Streptococcus sp. and Leptotrichia buccalis, showed similar relative abundance between periodontitis patients and healthy controls in saliva and supragingival plaque. The third OTU (OTU0288), which was highly homologous with Prevotella sp. showed different abundance between periodontitis and healthy samples , which were closely related to ples see  online.Firmicutes, followed by Bacteroidetes and Proteobacteria. However, in both supra and subgingival plaque, the top three phyla in abundance were Bacteroidetes, Firmicutes and Fusobacteria   was performed for saliva and supragingival plaque samples from periodontitis and healthy individuals. At the first step of RDA, health and periodontitis were used as environmental variables and the relative abundances of all OTUs in saliva or supragingival plaque were used as species variables. The Monte Carlo Permutation Procedure (MCPP) showed that the difference of microbiota between periodontitis and health in saliva or supragingival plaque was significant (= 0.001) . We iden= 0.001) . We idenPorphyromonas gingivalis, Porphyromonas endodontalis, Tannerella forsythia, Prevotella sp., Eubacterium nodatum, Filifactor alocis, and Peptostreptococcus stomatis. The other three OTUs had lower similarities (<97%) with existing hit taxa in the database, suggesting that they are likely novel phylotypes . Notably, strong positive correlation  was observed between OTU0263 and the remaining seven OTUs  which were closely related to P. gingivalis, P. endodontalis, T. forsythia, E. nodatum, Fretibacterium sp., Lachnospiraceae [G-8] sp. and Peptostreptococcaceae [XI][G-4] sp., seemed to form a F. alocis-centered bacterial cluster with co-occurrence relationships in the human oral cavity. This F. alocis-centered co-occurrence group collectively represented 6.28%, 5.49% and 29.11% of the oral microbiota in P-Sal, P-Sup and P-Sub, respectively.Based on the 21 periodontitis-relevant species, we investigated the correlations of each pair of them. Spearman's correlation coefficients were assessed based on the normalized relative abundances of the 21 periodontitis-associated phylotypes using all 150 oral samples from the three distinct oral niches of the two cohorts. Interestingly, eight OTUs displayed high correlation coefficients with each other  curves in which test sensitivity is plotted against 1-specificity or false positive rate (FPR), to evaluate the performance of diagnostic tests. Area under the ROC curve, cut-off point, sensitivity, specificity, likelihood ratio and P values were calculated and compared. In saliva, the area under the ROC curve of the co-occurrence group was the highest of all the tested phylotypes (0.817), implying that this cluster has the highest value for periodontitis diagnosis  and 1.58% in supragingival plaque , indicating that a person who has the abundance of this cluster over 2.11% in saliva or over 1.58% in supragingival plaque would have a high risk of periodontitis.To assess its performance in identifying the risk of periodontitis, the relative abundances of the iagnosis . The cutFor decades, efforts have been made to identify potential pathogens and elucidate their functions in the development of periodontitis. However, given the complexity of the oral microbiota, there is still no definitive evidence demonstrating how many different pathogens are involved in the polymicrobial disease and how these co-infecting species interact with each other in triggering the disease and contributing to its progression.Streptococcus, Leptotrichia and Actinomyces were commonly detected in all 150 samples with similar abundance in health and periodontitis. Such taxa are regarded as components of the core oral microbiota that adapt to the oral environment and provide structural support for the oral microbiota224In this investigation, saliva, supragingival plaque and subgingival plaque samples were used to characterize the oral microbiota. Steptococcus, Neisseria, Veillonella, Rothia, Granulicatella, Haemophilus and Gemella were predominant, which were previously reported as commensal bacteria colonizing the mucosal surfaces of mouth17Capnocytophaga, Leptotrichia and Corynebacterium. In subgingival plaque, obligate anaerobic bacteria such as Porphyromonas, Treponema, Tannerella, Fusobacterium and Filifactor were found as major members of this anaerobic niche. Thus, the different microbial distribution in diverse oral habitats may be mainly driven by the surface structure and oxygen condition of each microhabitatWe also found that each oral habitat harbored its distinct microbiome. In saliva, several genera including Via a microbiome-wide association study (MiWAS) strategy, 21 OTUs were found to be associated with periodontitis. MiWAS strategy combines high-throughput DNA-sequencing technology with multivariate statistical tools such as principal component analysis (PCA), principal coordinate analysis (PCoA) and redundancy analysis (RDA), to explore the structural variations across the entire human microbiome without any prior hypothesis, aiming to identify all relevant bacteria associated with diseases28P. gingivalis, T. forsythia and F. alocis, and were present in 93.3%, 90.0% and 92.2% of oral samples from periodontitis patients, respectively. These three OTUs accounted for 65.7% of the total abundance of the 21 OTUs in the three habitats of periodontitis patients. P. gingivalis, T. forsythia and F. alocis were frequently found to link with periodontitis in previous studies using subgingival plaque samples79101131P. gingivalis was the most widely studied periodontal pathogen, partially due to its high detection rate via culture methods4P. gingivalis could change the amount and the composition of the oral microbial symbionts, triggering inflammatory periodontal bone loss in a mouse modelDespite the difficulty of assigning an OTU to an exact species due to the short sequencing read lengths, OTU0054, OTU0204 and OTU0263 were found to be closely related to the putative periodontopathic bacteria P. endodontalis (OTU0096), E. nodatum (OTU0177), Peptostreptococcus spp. , Prevotella spp. , Lachnospiraceae [G-8] sp. (OTU0308), Bacteroidetes [G-3] sp. (OTU0304), Fretibacterium sp. (OTU0223), Johnsonella sp. (OTU0287), Leptotrichiaceae [G-1] sp. (OTU0146) and Mollicutes [G-2] sp. (OTU0309). These 14 phylotypes were previously reported to be associated with periodontitis in investigations based only on subgingival plaque samples10Actinomyces cardiffensis, a Gram-positive facultative anaerobic rod. This bacterium can be isolated from diverse human clinical sources such as pleural fluid, brain, jaw, pericolic and ear abscesses36In addition, we identified 18 other OTUs significantly associated with this disease, including OTUs that were closely related to lis OTU006, E. nodin vitro or animal experiments.For a polymicrobial disease, identification of potential pathogens is only the first step; delineating the species-species interactions of multiple pathogens is essential for understanding the microbial etiology of the disease. In the present study, we examined the pairwise correlation coefficient among the 21 potential periodontal pathogens identified, according to their abundance in all 150 oral samples from three distinct oral niches of the two human cohorts. A bacterial group composed of eight phylotypes showed a robust co-occurrence pattern. Their abundance varied among different oral samples with the same tendency, increasing together in three habitats of periodontitis patients and decreasing together in two habitats of healthy individuals. Our finding implied there might be synergistic interactions among them, which should be confirmed by further in vitro studies. Grenier demonstrated that P. gingivalis can produce isobutyric acid to stimulate the growth of T. denticola and metabolize the succinate produced by T. denticola to promote its own growthF. nucleatum can maintain a more neutral pH in the microenvironment to protect acid-sensitive species such as P. gingivalis against acid attackP. gingivalisP. gingivalis to invade human gingival epithelial cell was enhanced when co-cultured with F. nucleatumThe synergistic interactions between some cultivable indigenous oral pathogens have been revealed previously by F. alocis showed strong positive correlations with seven other members (R > 0.6 for each pair), thus seemed to be the center of this bacteria cluster. F. alocis is a Gram-positive obligate anaerobic rod, which was recently identified as one of the marker organisms for periodontal diseases using culture-independent approaches, due to its higher prevalence and abundance in periodontal disease sites compared with healthy sites71042P. gingivalisF. alocis is relatively resistant to oxidative stress and possesses several virulence factors, such as sialoglycoproteases and proteases, which may partially contribute to its colonization and survival in the periodontal pocket. In addition, in vitro experiments showed that F. alocis could induce apoptosis in gingival epithelial cells (GECs) and stimulate pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α secreted by GECsF. alocis may be an important pathogen in the formation of subgingival biofilm and the onset of periodontal diseases. Previous studies also showed that F. alocis could synergistically interact with other periodontal pathogens in vitro. For example, F. alocis could enhance the biofilm formation and the invasion of epithelial cells by P. gingivalis during cocultureF. alocis was also found accumulated around F. nucleatum-abundant regions and showed mutualistic growth with F. nucleatum in an in vitro community development modelF. alocis may similarly enhance the growth or virulence of other pathogens in the co-occurrence group, promoting the development of periodontitis.In the co-occurrence pattern we identified, a phylotype closely related to P. gingivalis, P. endodontalis, T. forsythia, E. nodatum, Fretibacterium sp., Lachnospiraceae [G-8] sp. and Peptostreptococcaceae [XI][G-4] sp. Each of these bacteria has been previously identified as associated with periodontitis in different studies by culture or culture-independent approaches591147P. gingivalis and T. forsythia were the only two microorganisms which were reported to be synergistic in previous studies. For example, in vitro experiment showed that P. gingivalis or its outer membrane vesicles could enhance the attachment and invasion of T. forsythia to epithelial cellset al. also reported that the co-infection of P. gingivalis and T. forsythia induced greater abscess size than did mono-infection of each bacterium in a murine modelThe remaining seven members of the co-occurrence group include F. alocis-centered co-occurrence group potentially has a high diagnostic value for periodontitis, especially in saliva where the cluster showed the largest area under the ROC curve and high sensitivity and specificity to discriminate periodontitis from healthy samples . Saliva is an abundant oral biofluid that can be collected noninvasively. Previous reports have revealed that oral bacteria such as A. actinomycetemcomitans, C. rectus, P. gingivalis, P. intermedia, P. nigrescens, P. micros, T. denticola, T. forsythia, F. nucleatum and T. socranskii are detectable and enriched in saliva from periodontitis patients, and therefore could be useful salivary biomarkers of periodontitis31F. alocis-centered co-occurrence group was higher than any single periodontitis-related phylotype in saliva, reinforcing the significant role of this group in periodontitis progression and providing a potential indicator to identify high-risk periodontitis patients.The Streptococcus, Leptotrichia and Actinomyces as a common core oral microbiota, and each habitat also harbors its distinct microbial members. Twenty-one species-level OTUs were identified as periodontitis-associated phylotypes, including a F. alocis-centered co-occurrence group consisting of eight phylotypes that were positively correlated with each other in various habitats of the oral cavity. The co-occurrence pattern of those potential pathogens underlines their ecological relationship relevant to disease progression. The findings from this study may serve as an ecological framework to facilitate not only the understanding of the complex etiology of periodontitis, but also the development of a new periodontal diagnoses and therapies targeting this co-occurrence group.In summary, we characterized the microbiota of three main oral habitats in human subjects with and without periodontitis. Saliva, supragingival plaque and subgingival plaque harbor A total of 30 periodontitis patients, aged 29–67 years, were recruited from the department of periodontology in Ninth People's Hospital . These patients took no periodontal treatment in the 3 months before sample collection. Thirty periodontally healthy volunteers, aged 21–55 years, were selected as controls. Inclusion criteria for all participants included no pregnancy or any systemic diseases, no antibiotic therapy within the past 3 months, and at least 20 teeth in the oral cavity excluding third molars. Every periodontitis patient had, in each quadrant, at least one posterior tooth with pocket depth ≥4 mm and attachment loss ≥2 mm, whereas healthy controls exhibited no site with pocket depth >3 mm or attachment loss ≥2 mm and displayed no clinical gingival inflammation . The baseline clinical characteristics of all individuals are showed in All the participants were instructed to refrain from eating, drinking and any oral hygiene practice for at least 3 h before sampling. After rinsing mouth with water, approximately 1 ml non-stimulated saliva was collected and transferred into 1.5 ml sterile centrifuge tubes. The selected teeth (four teeth with the highest level of pocket depth from four quadrants) were then isolated using cotton rolls and gentle air-drying. Supragingival plaque was collected first, followed by subgingival plaque from the bottom of the periodontal pocket of the same teeth using sterile subgingival curettes. The pooled supragingival plaque and the pooled subgingival plaque from each subject were separately stored in 1.5 ml sterile centrifuge tubes containing 1 ml sterile phosphate-buffered saline (PBS). All samples were immediately frozen at −80°C until DNA extraction. In total, 150 samples were collected and divided into five groups according to the sampling habitats and populations: H-Sal , H-Sup , P-Sal , P-Sup , P-Sub . Subgingival plaque samples were not collected from healthy controls because there is no distinct periodontal pocket formed in healthy gingival tissue.CCTACGGGAGGCAGCAG-3′ and the reverse primer 5′-NNNNNNNNATTACCGCGGCTGCT-3′, marked at the 5′ end with a sample-unique 8-base barcode, was used to amplify the V3 region of 16S ribosomal RNA gene from each oral sample. PCR amplification, pyrosequencing of the PCR amplicons, and quality control of raw data were performed as described previouslyOral samples were washed twice with PBS and underwent two cycles of freeze-thawing at −80°C for 10 min and 60°C for 2 min. Genomic DNA was then extracted from each oral sample using bead-beating extraction and phenol-chloroform purification, as described previously28http://rdp.cme.msu.edu/classifier/classifier.jsp). The data of RDP taxonomy was used in LEfSe to discover the taxa differentiating the microbial communities specific to different oral habitatsAll high-quality pyrosequencing reads were aligned against the Greengenes database using the nearest alignment space termination (NAST) algorithmhttp://www.uga.edu/strata/software), Shannon diversity index (H') (R package 2.12.0), as well as Simpson's diversity index (1-D) and Chao1 estimate were calculated (QIIME). The most abundant sequence of each OTU was selected, imported into the ARB and inserted into a pre-established phylogenetic tree of the full-length 16S rRNA gene sequences to generate a phylogenetic tree. The phylogenetic tree, together with sequence abundance data, was then used for online Fast UniFrac analysis based on a weighted metric (http://unifrac.colorado.edu/). The most abundant sequence of each OTU was BLAST searched against the Human Oral Microbiome Database (HOMD) to find out the nearest neighbor of the OTU (http://www.homd.org/modules.php?op=modload&name=RNAblast&file=index&link=upload). Relative abundances of OTUs were used for correlation analysis via Matlab R2010a . Redundancy analysis (RDA) was performed using CANOCO for Windows 4.5 . ROC curves were constructed to determine the diagnostic values for periodontitis of key periodontitis-associated phylotypes or co-occurrence group via SPSS Statistics 21 . The area under the ROC curve is the most commonly used index to evaluate the diagnostic accuracy, with values close to 1.0 indicating high diagnostic accuracy. The points displaying the largest Youden's index (sensitivity + specificity - 1) were defined as the optimal cut-off points.Meanwhile, those representative sequences after CD-hit were imported into the ARB to construct a neighbor-joining tree. Operational taxonomic unit (OTU) was delineated at 96% similarity level with DOTURConceived and designed the experiments: X.P., Z.T. and L.Z. Performed the experiments: H.C. Analyzed the data: H.C. Contributed reagents/materials/analysis tools: Z.T., Y.L., Z.Q., M.Z. and G.W. Wrote the paper: H.C. and X.P. Revised the manuscript: X.P., L.Z. and L.B.These sequence data have been uploaded to the Sequence Read Archive database under the accession number SRP050141.Supplementary information


Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.Shigatoxigenic stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.The whole genomes of three STEC/ETEC strains possessing both E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.This study shows that pathogroup-associated virulence genes of different  Escherichia coli (STEC) and other diarrheagenic E. coli (DEC) cause diarrheal disease in humans [stx1 and stx2), which are responsible for the toxic effects in the host.Shigatoxigenic n humans . STEC caE. coli (ETEC) cause watery diarrhea by producing heat-labile LT (encoded by elt) and/or heat-stable ST (encoded by estIa porcine variant and/or estIb human variant) enterotoxin. Enteropathogenic E. coli (EPEC) produces characteristic histopathology known as attaching and effacing on intestinal cells. Enteroinvasive E. coli (EIEC) is associated with invasive, bloody diarrhea resembling that caused by Shigella spp. Enteroaggregative E. coli (EAEC) harbors the mechanism for aggregative-adherence pattern mediated by aggregative adhesive fimbriae. EAEC is increasingly recognized as a diarrheal pathogen in developing countries.Several other DEC pathogroups have been established based on the pathogroup-associated virulence traits . Enterotintermediate or hybrid pathogroups [STEC and other DECs are able to acquire virulence genes via horizontal gene transfer from other pathogroups leading to the development of hogroups –3. A hybhogroups . Hybridshogroups –7, some hogroups  and fromhogroups .E. coli is a genetically versatile species. Strains within a single pathogroup can originate from different genetical backgrounds [stx-phages multiple times [E. coli strains belonging to different phylogenetic lineages can independently evolve into enterohemorrhagic form of STEC by acquiring phages and other integrative elements, such as LEE, essential for the virulence properties [elt or est genes may be enough to make an ETEC strain [E. coli K-12 strain MG1655 suggesting that the main event in the emergence of ETEC from E. coli is the acquisition of virulence plasmids carrying elt or est [kgrounds –13. Amonle times . In addioperties . Also thoperties . Multi lC strain . In addit or est .E. coli strains have not been studied widely. An exception is the German outbreak strain EAEC/STEC O104:H4, which was shown to form a distinct clade with other O104:H4 strains among EAEC and E. coli indicating that the outbreak strain has the chromosomal backbone similar to EAEC O104:H4 group [Genomics and phylogeny of hybrid H4 group . In a reH4 group . This suE. coli and to define the similarities and differences in their gene contents and virulence properties related to other DEC pathogroups. We used whole genome sequencing and whole genome mapping for comparative genomics between the STEC/ETEC genomes and the reference genomes of pathogenic and commensal E. coli and Shigella spp. It is crucial to understand the phylogeny of pathogenic bacteria to evaluate how they have evolved and to monitor the emergence of potential new pathogens.In the present study, we investigated human and bovine STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli strains possessing both STEC- and ETEC-associated virulence genes stx1 or stx2 and estIa were sequenced. The strains have been described in our previous studies [-) was isolated from a 1.9-year-old infant with HUS in Finland and the strain IH57218 (serotype O2:H27) was isolated from a 7.3-year-old child with diarrhea in Finland [Shigella spp. strains were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank) (The whole genomes of three  studies –9. The sgenbank) .Genomic DNA was extracted using QIAGEN Genomic Tip 100/G  according to the manufacturer’s instructions. After the extraction, the intactness of the genomic DNA was verified by agarose gel electrophoresis and the quantity of the genomic DNA was measured by Qubit 2.0 Fluorometer .Sequencing libraries were constructed according to the manufacturer’s  protocol. Sequencing was done on the PacBio RS instrument (Pacific Biosciences) with P4/C2 chemistry.de novo assembly, the HGAP pre-assembly workflow was used to generate long and highly accurate sequences. This was accomplished by mapping single pass reads to seed reads, which represent the longest portion of the read length distribution. Subsequently, a consensus sequence of the mapped reads was generated resulting in long and highly accurate fragments of the target genome. We further pruned reads from this pre-assembly pipeline that were < 3500bp. The pre-assembled reads were output to FASTQ format and further error corrected using the PacBioToCA utility in Celera Assembler (CA 7.0). The reads were then assembled with the CA 7.0 assembler using the BOGART algorithm with mer size of 25. Scaffolding was carried out using the CGW algorithm of the CA 7.0 assembler.The data collected from the PacBio RS instrument were processed and filtered using the single molecule real-time (SMRT) analysis software suite (Pacific Biosciences). Data were filtered by read quality (> 0.75) and read length (> 1000 bp). When processing continuous long read (CLR) data, raw reads from the SMRT Cells were split on adapter sequence resulting in ≥ 1 subread or CLR per zero-mode waveguides (ZMW). For SMRT The final number of contigs per genome after the assembly was 10 for IH53473, 17 for IH57218, and 43 for FE95160. The three draft genomes were uploaded into Galaxy/CRS4 (Orione)  for autoNcoI restriction enzyme. Whole genome maps were produced using Argus Optical Mapping System  as previously described [NcoI in silico and the contigs were aligned against the map using MapSolver 3.2.0 software (OpGen Inc.).Whole genome mapping  was used to correct the order of contigs and to detect any misassemblies. For this, the chromosomal DNA was digested using escribed . To compE. coli virulence genes . Primer sequences for ETEC colonization factors CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS12, CS13, CS14, CS15, CS17, CS17-19, CS20, CS21, and CS22 were obtained from previously published articles [stx1 and seven stx2 subtypes were obtained from the published PCR protocol [stx1 and stx2 subtyping was performed to compare in silico typing to the previous stx1 and stx2 subtyping PCR results [The genomes were screened for a broad spectrum of known and characterized ce genes  using Riarticles . Primer protocol . stx1 aadk, fumC, gyrB, icd, mdh, purA, recA, used in the published protocol for E. coli MLST [E. coli MLST application in Ridom SeqSphere+ program (Ridom GmbH) to determine MLST sequence types (ST) of the STEC/ETEC genomes and reference genomes.The allelic profiles of the seven genes, oli MLST , were exShigella spp. were determined in silico using phylotyping primers for the E. coli phylogroups A, B1, B2, C, D, E, F, and cryptic clades I-V [Phylogroups of the STEC/ETEC genomes and reference genomes except ades I-V –28 and Gin silico serotyping results. The genes responsible for the expression of O- and H-antigens were detected from the genomes. The primers and probes to detect the respective O- and H-sequences were previously published [The three STEC/ETEC strains were previously serotyped using the traditional O- and H-antigen agglutination –9. We noublished –30. The stx-phages were determined manually. The stx genes were located in the assembled contigs. Starting from the stx gene, the sequence upstream and downstream was screened for the phage-related genes using the BLAST tool [PHAST tool (phast.wishartlab.com)  was usedAST tool  and GeneProduction and titers of Stx and ST were determined. Stx was tested on the Vero cell assay at Statens Serum Institut . ST was determined using the GM1-ELISA method as previously described –34. The estIa, hlyA, espP, and astA in the contigs of the draft genomes. PROKKA annotation reports were also utilized in the survey of the possible plasmid sequences.PlasmidFinder 1.2  was usedE. coli and Shigella spp. reference genomes, both completed and draft genomes . Whole genome maps were also used to generate phylogenetic tree where the three STEC/ETEC whole genome maps were compared with in silico NcoI restricted maps generated from completed reference chromosomal sequences . No other SPATEs were detected. IH53473 possessed also genes irp1, irp2, and fyuA encoding yersiniabactin biosynthetic proteins and a receptor. IH53473 was positive for LEE pathogenicity island, which contains the gene eae encoding intimin, espA and espD encoding translocators, and escV, espF, and espH encoding type III secretion system structure and effector proteins. The human strain IH57218 was positive for Shigella enterotoxin 2. The bovine strain FE95160 was positive for astA encoding EAEC heat-stable enterotoxin I and aai pathogenicity island encoding type VI secretion system.All three STEC/ETEC strains harbored multiple virulence genes . All theeaeH, putative outer membrane autotransporter adhesin gene yfaL, type 1 fimbria gene fimH, and common pilus subunit gene ecpA  and was assigned with a new ST number ST4123.The STs of the human strains were of the previously established types: ST330 for IH53473 and ST10 for IH57218. The ST of the bovine strain FE95160 was novel. It was submitted to the E. coli phylogroup A. The bovine strain belonged to the cryptic clade I.Both human strains belonged to the -/non-motile had primer and probe binding sites for H33. FE95160, which had previously been typed as H2 had primer and probe binding sites for H25. H-typing result of strain IH57218 was consistent with the previous result H27.O-grouping results were consistent with the previous results: IH53473 O101, IH57218 O2, and FE95160 O2. However, H-typing results were different in two strains. IH53473, which had previously been typed as HFor strain IH53473, 16 prophage regions were identified, of which nine regions were intact, three regions were incomplete, and four regions were questionable. For strain IH57218, 17 prophage regions were identified, of which 12 regions were intact, and five regions were incomplete. For strain FE95169, 10 prophage regions were identified, of which seven regions were intact, and three regions were incomplete.stx2a-phages in IH53473 and IH5728 were identified in wrbA locus. Both phages were similar to phage P13374 (accession no. HE664024) by BLAST search. The integration site for the stx1a-phage in strain FE95160 was between the genes ybhC and ybhB. The phage remained unidentified as no hits were found by BLAST search.The integration sites for the All the three STEC/ETEC strains expressed Stx with titers of 1:100,000 dilution. IH57218 and FE95160 produced STIa. The STIa titers were 168 ng/ml for IH57218, 40 ng/ml for FE95160, and 253 ng/ml for ETEC H10407 control strain. IH53473 did not produce STIa.estIa, hlyA, and espP were placed in the same contig as IncFII(29). IH57218 had one plasmid replicon: IncFII. The plasmid-associated genes estIa and hlyA were placed in a different contig than IncFII. FE95160 had two plasmid replicons: IncFII(pSE11), and IncFIB(AP001918). The plasmid-associated genes estIa, hlyA, and astA were placed in the same contig as IncFIB(AP001918). According to the PROKKA annotation reports of IH53473 and FE95160, several plasmid-associated genes, such as RepFIB replication protein A, plasmid partitioning protein B, and plasmid stability protein, were located in the same contigs as the virulence genes estIa, hlyA, espP, and astA. According to the PROKKA annotation report of IH57218, several plasmid-associated genes were also found in the same contig with estIa and hlyA. However, no origin of plasmid replication was present in that contig but it was located in another one.PlasmidFinder indicated several plasmid replicon sequences of known Inc groups in the STEC/ETEC genomes. IH53473 had three plasmid replicons: IncQ2, IncFII(29), and IncXI. The plasmid-associated genes E. coli and Shigella spp. genome sequences using cgMLST and sequence alignment. The analysis showed that different E. coli pathogroups are inter-mixed , which is prevalent in several EAEC isolates but rarely detected in other DECs [espP, was also detected in IH53473. SPATE is a family of extracellular proteases produced by the species belonging to Enterobacteriaceae and they have an impact on mucosal damage and colonization [astA encoding EAEC heat-stable enterotoxin I. Unlike the toxin name indicates, astA can be harbored by strains of STEC, EAEC, EPEC, ETEC, and EIEC pathogroups [E. coli [aai pathogenicity island encoding putative type VI secretion system, which was also detected in the German outbreak strain EAEC/STEC O104:H4 [The varying combination of multiple virulence genes harbored by the STEC/ETEC hybrids showed that the strains were a mixture of several different thogroup . The humher DECs ,38. HPI nization . The Gernization . The bovhogroups  and even[E. coli . FE95160 O104:H4 .eaeH, yfaL, ecpA, and fimH associated with adhesion [ecpA and fimH have been associated with ETEC pathogroup [Shigella enterotoxin 2, which can increase virulence [shiA encoding shikimate transporter in a pathogenicity island involved in the suppression of host inflammatory response [All the STEC/ETEC strains possessed some of the genes adhesion ,16,42–43thogroup . It is nthogroup . Thus, tirulence . All STEresponse .E. coli strains. On the contrary, the bovine strain FE95160 shares similar genetic background with two STEC genomes of bovine origin and they form a remote cluster in the phylogenetic tree. The phylogeny inferred from whole genome map similarity clustering supports these observations. Also the whole genome map comparison between the three STEC/ETEC strains is consistent with the remote phylogenetic position of the bovine strain since the human strains were shown to share more genetic elements with each other than with the bovine strain.The phylogeny inferred from cgMLST and sequence alignment demonstrates that our STEC/ETEC hybrid strains do not form a single cluster. The phylogenetic placement of the human STEC/ETEC strains indicates a common ancestor with certain ETEC, STEC, EAEC, laboratory-adapted and commensal The previous studies have shown that both STEC and ETEC pathogroups are genetically versatile ,14–15. OestIb and stx2e genes, and with ETEC O147 UMNF18 [estIa, estIb and stx2e genes. Our bovine STEC/ETEC clustered with STEC O2:H25 7v [estIa and stx2g genes. The other two STEC/ETEC hybrids from Steyert et al. study [stx-phages.Even though all our STEC/ETEC strains did not cluster together, we found evidence that STEC/ETEC hybrid strains may have similarities in their chromosomal background. In our study, we combined our data with the previously sequenced five STEC/ETEC hybrid genomes in cgMLST ,37. Our E. coli MLST ST10, which includes our human STEC/ETEC strain IH57218, is common among the ETEC strains from human origin [estIa gene [estIa gene. IH53473, which was shown to be SPATE espP positive, clustered together with an EAEC genome in cgMLST based UPGMA tree. EAEC often harbor several SPATEs, and espP is class I cytotoxic SPATE [n origin . InterestIa gene , as is tic SPATE . IH53473stx2a, stx2c, or stx2dact show generally higher cell cytotoxicity compared to stx2b, stx2e, or stx2g [stx2a were isolated from patients with HUS or diarrhea. IH53473 also carried eae. Clinically relevant STEC, and especially eae positive STEC, have shown high cytotoxicity levels compared to food isolates, which have shown more diverse cytotoxicity levels [estIa gene.Two of the STEC/ETEC strains were able to produce both Stx and STIa. The ability to produce both toxins may result in increased virulence. All three STEC/ETEC strains had a very high Stx titer of 1:100,000 dilution. There was no difference in Stx cytotoxicity between the human and bovine STEC/ETEC strains. Human isolates possessing r stx2g . Our humIn silico O-grouping and H-typing may be used to replace agglutination-based serotyping. In the present study, O-grouping results were consistent with the previous results obtained by antiserum agglutination [in silico method showed primer and probe binding sites for H25. The results may be due to the fact that the H-agglutination schema [In silico typing seems to be a good choice especially for non-motile strains which cannot be typed by agglutination due to the lack of the expression of H-antigen.tination –9. Howevn schema  has onlyestIa and astA may be plasmid- or chromosome-associated [estIa, hlyA, espP, and astA in the genomes of IH53473 and FE95160. This may indicate that the estIa genes of IH53473 and FE95160 and astA gene of FE95160 are located on plasmids rather than the chromosome. The assembly pipeline favors the assembly of plasmid sequences in separate contigs. Also estIa in IH57218 genome may be plasmid-associated. Even though the plasmid replicon sequence was detected in another contig than estIa, this may be due to fragmentation of plasmid sequence in two separate contigs during the assembly.The genes sociated . The locstx-phage integration sites in the STEC/ETEC genomes were the usual ones. In the IH53473 and IH57218 genomes stx-phage interrupted the wrbA gene. The gene wrbA encoding tryptophan repressor binging protein is a common integration site for the stx2 phages [stx-phage integration site between the genes ybhC (predicted pectinesterase) and ybhB (predicted kinase inhibitor). We screened the E. coli reference genomes for similar cases and noticed that also E. coli UMN026 (accession no. NC_011751.1) and E. coli EDL933 (accession no. NC_002655.2) possess phages on this site.All our STEC/ETEC strains possessed several prophage regions in their genomes. It is typical of STEC genomes to harbor prophages and other integrative elements . The ide2 phages . The FE9E. coli and development of STEC variants [E. coli strains [est [estIa in the STEC/ETEC hybrids may be associated with plasmids. Nevertheless, it is not surprising that both ETEC and STEC strains can arise in different phylogenetic groups and that they do not necessarily have a clonal lineage. Some commensal strains may have pathogenic potential since certain parts of their genomes may act as genetic repositories for virulence factors [The Stx-phages and their ability to transfer genes horizontally play an important role in the evolution of variants . Also th strains . However strains . ETEC plins [est ,51. Someins [est , as was  factors . AcquisiE. coli infections.The comparative genomics of the STEC/ETEC hybrid strains showed that STEC- and ETEC-associated virulence genes can co-exists in strains originating from different phylogenetic lineages. Whole genome sequencing techniques enable fast typing and possibility to screen several genetic markers simultaneously making it easy to detect virulence genes associated with several pathogroups. An infection with a hybrid pathogenic strain may result in more severe disease in a patient. These strains may also have increased spreading potential. Therefore, their emergence should be taken into account in modern strain typing and in epidemiological surveillance of S1 Fig(A) Comparison between IH53473 and IH57218, (B) comparison between IH53473 and FE95160, and (C) comparison between IH57218 and FE95160.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


In Japan, all trainee physicians must begin clinical practice in a standardized, mandatory junior residency program, which encompasses the first two years of post-graduate medical training (PGY1 – PGY2). Implemented in 2004 to foster primary care skills, the comprehensive rotation program (CRP) requires junior residents to spend 14 months rotating through a comprehensive array of clinical departments including internal medicine, surgery, anesthesiology, obstetrics-gynecology (OBGYN), pediatrics, psychiatry, and rural medicine. In 2010, Japan’s health ministry relaxed this curricular requirement, allowing training programs to offer a limited rotation program (LRP), in which core departments constitute 10 months of training, with electives geared towards residents’ choice of career specialty comprising the remaining 14 months. The effectiveness of primary care skill acquisition during early training warrants evaluation. This study assesses self-reported confidence with clinical competencies, as well as case experience, between residents in CRP versus LRP curricula.A nation-wide cross-sectional study of all PGY2 physicians in Japan was conducted in March 2011. Primary outcomes were self-report confidence for 98 clinical competency items, and number of cases experienced for 85 common diseases. We compared confidence scores and case experience between residents in CRP and LRP programs, adjusting for parameters relevant to training.Among 7506 PGY2 residents, 5052 replied to the survey (67.3%). Of 98 clinical competency items, CRP residents reported higher confidence in 12 items compared to those in an LRP curriculum, 10 of which remained significantly higher after adjustment. CRP trainees reported lower confidence scores in none of the items. Out of 85 diseases, LRP residents reported less experience with 11 diseases. CRP trainees reported lower case experience with one disease, though this did not remain significant on adjusted analysis. Confidence and case experience with OBGYN- and pediatrics-related items were particularly low among LRP trainees.Residents in the specialty-oriented LRP curriculum showed less confidence and less case experience compared to peers training in the broader CRP residency curriculum. In order to foster competence in independent primary care practice, junior residency programs requiring experience in a breadth of core departments should continue to be mandated to ensure adequate primary care skills. In the rapidly specializing health care arena, the training of competent generalists remains a cornerstone of optimal population-level health as measured by a variety of outcomes in many countries . DespiteTo adequately meet demand for generalist practitioners, it is crucial that young physicians receive adequate exposure to the full breadth of general practice. However, curricular structures that best meet this need, balancing core generalist competencies with specialized knowledge and skills, remain the subject of ongoing discussions amongst which curricular redesigns are not uncommon. To this end, in 2007, the Alliance for Academic Internal Medicine Education Redesign Task Force, sought to formalize a core set of knowledge, skills, and attitudes, proficiency in which should be maintained throughout a career, regardless of practice in a specialty or general medicine .Formal residency training for recent medical school graduates was established more than 60 years ago in Japan. However, rules governing post-graduate training previously allowed the vast majority of young doctors to immediately begin training in their specialty department of choice, without mandating any period of time in either a general medicine or surgical curriculum. Nonetheless, due to the historical absence of formally trained primary care physicians in Japan, specialist trainees are expected to acquire skills needed to treat a wide variety of diseases common to primary care . To bettAs of 2012, two different types of rotation programs exist in Japan, giving us a unique opportunity, one not found in other countries, to directly assess the efficacy of generalist training using two competing curricula offering a difference in breadth of training. Differences in training effectiveness of these two systems have not yet been assessed. The purpose of this study is to investigate PGY2 physicians’ self-reported confidence with knowledge, skills, and experience of diseases useful to primary care, and to compare these between residents trained in two different rotation curricula. We hypothesize that residents training in a more comprehensive program will be more confident and more experienced in regards to a variety of conditions seen in general practice compared to those residents’ opting for more limited curricula.In Japan, specialty training is most often undertaken at university hospitals; 70% of residents in 2001 trained in a university hospital program, with only 30% belonging to an unaffiliated community hospital .st - and 2nd-year (“junior”) residents to rotate through a standardized set of core departments comprised as follows: internal medicine subspecialties (6 months), surgery (3 months), anesthesiology (1 month), obstetrics-gynecology , pediatrics (1 month), psychiatry (1 month), and rural/community medicine (1 month); the additional 10 months are generally spent in a variety of hospital-specific required rotations or career-oriented electives. As opposed to medical school clerkships in which Japanese students have limited patient contact [Adult primary care residency curricula  are relatively recent phenomena in Japan and are not widely offered as formal training tracks. Recognizing the need for adequate primary care training, the Ministry of Labor, Health, and Welfare (MLHW) of Japan implemented a mandatory junior residency program in 2004 with the purpose of fostering residents’ experience and skill in primary care, as well as enhancing professionalism and patient-centered practice. The CRP curriculum requires all 1 contact ,9, the C contact . HistoriA nation-wide cross-sectional study of graduating PGY2 residents was conducted in Japan in March 2011, the end of the Japanese academic year. Participants were informed in writing prior to starting the questionnaire that survey completion implied consent for use of the data for research purposes. Participation was voluntary and uncompensated; there was no penalty for non-participation. In addition to residents’ baseline demographic data, survey data included type of training curriculum (CRP versus LRP) and parameters relevant to medical training, such as institutional type (university- versus community-based program). First implemented at the behest of the Health Ministry of Japan in 2008, the survey methods have been reported previously -12. BrieMain outcomes were self-reported confidence for 98 clinical competency items and number of cases experienced for 85 medical conditions. Confidence was recorded on a 4-point Likert scale as follows: 1 = very confident to perform independently; 2 = fairly confident to perform independently; 3 = no confidence to perform independently; and 4 = cannot perform. Number of cases experienced for 85 diseases commonly encountered during 2 years in residency training were recorded as follows: 1 = no cases, 2 = 1-5 cases, 3 = 6-10 cases, or 4 = ≥11 cases. Clinical confidence was dichotomized into ≤2 points versus ≥3 points for analysis, while case experience scores were dichotomized by ≥1 case versus no case experience. Case experience was further categorized into ≤2 points versus ≥3 points for multivariate logistic regression analysis, anticipating that many residents experienced at least 1 case for most diseases. All clinical competency items and diseases are detailed in the appendix (Appendix 1 and 2). Surveys were distributed via the administrative offices of individual residency programs. Participation in the survey was voluntary and residents received no compensation for participation.Anonymized data were collected and descriptive statistics were used to explore the data. Chi-squared tests were used to compare high confidence in clinical ability by comparing the percent of residents reporting a confidence score of 1 or 2 in CRP versus LRP programs. Similarly, Chi-squared tests were used to compare the percent of residents reporting at least one case experienced of each clinical survey item. Considering that age and gender have previously been reported to influence work and learning experience of Japanese residents , and thaTable For clinical competency items, LRP residents reported lower confidence scores in 12 items Table .Regarding experience with diseases commonly encountered in primary care, LRP residents reported less experience with 11 diseases; OBGYN- and pediatrics-related items and cases were found to be especially low Table . After aDespite conducting the survey at the end of the academic year (month 24 of a required 2-year curriculum), there were 775 residents (20.2%) in LRPs and 95 (7.9%) in CRPs who, for unclear reasons, reported ≤20 months of service (“non-compliers”). To address this, we conducted a sensitivity analysis in which non-compliers were excluded; we then conducted the same analysis for both competency and case experience. The results remained unchanged, suggesting that the impact of non-compliers is likely small.This nationwide study evaluated self-reported clinical competency and number of cases experienced between residents in two different curricular programs completing their 2-year junior training requirements. This survey is the first to compare two different post-graduate training systems on a national level, yielding several important results.First, proportionally more residents in university-based residency programs reported being trained in an LRP curriculum compared to residents at community hospitals. Community hospitals, typically offering a wider spectrum of primary care health services, have historically tended to offer broader rotation programs in order to meet unique community needs, such as those of rural and underserved areas. In contrast, university hospitals have historically focused on advanced, specialty-oriented care, as well as fostering the growth of basic science and translational research. Nonetheless, a large proportion of mid-career physicians from both university and community programs, regardless of specialty or geographic location, will leave their respective organizations in order to establish community-based primary care practices. This highlights the need for a modicum of robust generalist training for all physicians, regardless of training institution or practice location.While the dichotomy between general and specialty medicine exists in health systems outside of Japan, as evidenced by increasing resident shifts from generalist to specialist post-graduate training programs in the United States, the benefits of specialists receiving initial training in an accredited general curriculum has been a well-established educational paradigm in both the US and UK, as well as numerous other countries -16. In aSecond, LRP residents were found to be both less confident and less experienced in a number of clinical competency items and diseases compared to residents training in the broader CRP curriculum, even after adjusting for institutional parameters, gender, and age. Though the majority of items were comparable, that CRP trainees were never associated with less confidence or case experience is a compelling finding. One reason for this may be that significantly more community hospitals offer CRP curricula; because community hospitals focus more heavily on primary-oriented care, residents in CRP likely have more opportunity to experience care of undiagnosed patients, as well as patient care in a wide variety of medical departments. However, the association between CRP training and both higher confidence and case experience continued to be significant, even after adjustment for institutional type. It is reasonable to assume that, apart from the site of training, broader curricula that allow for more access to multiple departments do indeed lead to more effective post-graduate medical training.LRP residents’ reporting of areas of lower case experience and less clinical confidence appeared to be non-random and weighted towards basic care of relatively underserved populations in Japan. Suboptimal services included those for psychiatric patients (4 items) including diagnosis of depression, a fundamental skill in primary care. LRP residents also demonstrated lower confidence and less case experience in pediatrics (3 items) including viral infections and otoscopy, fundamental skills in general practice. Similarly, in OBGYN-related items (2 items), LRP residents reported less confidence with recognition and diagnosis of pregnancy. To put these findings in context, the large majority of items involving adult internal medicine were similar between CRP and LRP trainees. Albeit worrisome, these findings are unsurprising, as mandatory rotations through psychiatry, pediatrics, and obstetrics-gynecology were removed from LRP training requirements, thus limiting residents’ exposure to these items during emergency and community medicine rotations. While the argument may be made that pediatric and OB-GYN training is better restricted to those trainees specifically choosing careers in these fields, Japanese law and society place little restriction on subspecialists later returning to independent community medicine (“solo”) practices, of which women and children may comprise a substantial portion of the patient population. CRP training curricula may offer an additional safeguard to ensure that providers who do provide care to children and pregnant women have at least a modicum of training in these fields. This may be especially critical for Japan, which has experienced substantial shortages of pediatric and OB-GYN physicians over the last two decades ,18. ThisThough the strength of this study primarily lies in its large numbers and impressive response rate, there are several issues that warrant discussion. First, these competency data are based on self-reported confidence assessment, and self-reported number of cases experienced. Though objectively registered outcomes would be ideal, given the national-level scope of this study, it was not feasible to implement a third-party observer-driven study design. It should be noted that, while there is much interest in objective and standardized assessments of cases experienced and competency in post-graduate medical training, these are currently not routinely assessed in the majority of Asian, North American, or European programs. While an element of bias may be introduced in our study, we expect this to be largely non-differential in nature between CRP and LRP participants. Second, we categorized training curricula as either CRP or LRP based on residents’ self-reported time spent in departmental rotations. However, as noted, a sizeable number of residents, especially those in LRP programs, reported training for less than the required 24 months, suggesting that some programs may be non-complaint with either curricular strategy. This may indicate a policy-level need for ensuring that standardization in training requirements is followed uniformly. Currently, there is no universally-accepted accrediting organization in Japan to ensure compliance with residency training standards; our data suggests a need for further investigation regarding compliance with national training guidelines. Should such an organization be established, the monitoring of objectively registered outcome data may be more feasible and would warrant evaluation to corroborate our results.In addition, while many medical education systems around the world grapple with finding the optimal balance between specialty and general training, our results are most valid for the Japanese training context in which the survey was designed and implemented; country- and organization-specific needs and solutions addressing organizational structure, labor resources, work culture, as well as the economic and political dynamics of healthcare warrant careful consideration and thorough evaluation.Nonetheless, our data strongly suggests that broader training curricula may effectively enhance confidence in general care, and raises interesting avenues for future research. Specifically, longitudinal research to evaluate whether higher confidence translates into measurable improvements in skill and outcomes, and whether these improvements last into mid-career, are needed to fully clarify the impact of training models on large-scale health quality improvement.Residents in limited rotation programs reported both less confidence and less case experience on clinical competency items and diseases compared to their peers in broader comprehensive rotation programs. To acquire and maintain crucial generalists skills needed later in physicians’ careers, especially those involving pediatrics and gynecology, residency training programs should consider ensuring that all residents receive standardized training in a wide variety of core departments, with particular attention to the care of women and children.1. Elicit patients' interpretative model of health/care2. Systematic approach to patient medical history3. Non-verbal patient communication4. Gathering vital sign data5. Describing skin findings6. Diagnosis of arteriosclerosis by ophthalmoscopy7. Diagnosis of otitis by otoscopy8. Palpation of thyroid9. Palpation of apex beat10. Examination and assesment of heart sounds11. Examination and assesment of wheeze12. Examination and assesment of abdominal guarding13. Diagnosis of prostate abnormality by digital rectal examination14. Examination and assesment of early signs of pregnancy15. Diagnosis of genital abnormality on bimanual examination16. Examination and assessment of joint range of motion17. Examination and assesment of meningeal signs18. Examination and assesment of pediatric psychomotor developmental abnormalities19. Evaluation and assesment of diagnostic criteria for depression20. Diagnosis of bone fracture/dislocation/sprain21. Diagnosis of RBC and WBC casts on urinalysis22. Fecal occult blood testing and intepretation23. Blood gas analysis and interepretation24. Application and interepretation of leukocyte differential25. Application and interepretation of blood chemistry26. Application and interepretation of coagulation studies27. Application and interepretation of other routine blood exams28. Application and interepretation of immunologic blood testing.29. Analysis and intepretation of endocrinological examinations.30. Gram stain examination and intepretation.31. Cerebrospinal fluid examination and intepretation.32. ECG examination and intepretation of arrhythmia.33. Pulmonary function testing and diagnosis of pulmonary disease, including COPD.34. Biliary ultrasound testing and assessmnent of bile duct.35. Chest X-ray intepretation and dagnosis of silhouette sign.36. Abdominal X-ray intepretation and diagnosis of ileus.37. Chest CT intepretation and diagnosis of lung lesions.38. Brain MRI intepretation and diagnosis of cerebral infarction.39. Appropriate pre-operative handwashing.40. Appropriate venous blood sampling.41. Appropriate arterial blood sampling.42. Blood type cross-matching analysis and intepretation.43. Appropriate type and amount of IV for volume resuscitation.44. Lumbar puncture45. Urinary catheterization46. Appropriate understanding of use and side effects of antibiotics47. Performing local anesthesia and management of complications48. Incision and debridement49. Wound suturing50. Management of post-operative complications51. Management of pre-operative patient anxiety52. Cardiac massage53. Endotracheal intubation54. Initiation and maanagement of ventilator55. Cardiopulmonary resuscitation and AED use56. Assessment of severity of ER patients57. Diagnosis and management of shock58. Patient- and family-centered commnication of terminal cancer diagnoses59. Guidance and resource couselling for patients choosing home care.60. Palliative care team participation (including WHO cancer pain treatment).61. Consideration of both physical and psychosocial components of care.62. Provision of information regarding medical costs, social services, and family couselling resources.63. Taking appropriate informed consent.64. Appropriate consultation with supervising phyisicians and specialists.65. Provision of diabetic education.66. Knowledge of insurance points for routine exams.67. Understanding of role of social work and appropriate utilization.68. Smoking cessation69. Provision of patient education appropriate to knowledge and interest level70. Evaluation and assessment for home care.71. Utilization of community service resources and appropriate adjustment of treatment plans at discharge.72. Understanding of role of community services and social wealfare institutions and appropriate utilization.73. Evidence-based practice improvement including online searches.74. Patient presentation skills during hospital conferences.75. Patient charting (including discharge summaries) using the POS (Problem Oriented System) structure.76. Understanding of research design and appropriate use of scientific articles.77. Presentation at academic conferences.78. Understanding and use of appropriate statistical analyses.79. Understanding and implementation of patient safety paradigm.80. Adherance to patient safety protocols.81. Basic understanding of infectious disease control paradigms and protocols.82. Rapid assessement of geriatric audiovisual and cognitive function.83. Condunting of physical exams appropriate to geriatric patients' abilities.84. Geriatric management appropriate to physical, mental, and social functioning.85. Pediatric IV placement and phlebotomy.86. Management of physical and psychosocial issues among pediatric patients.87. Provision of appropriate explanations to pediatric patients.88. Pharmacologic management of common psychiatric diseases.89. Management of psychiatric pharmacotherapeutic complications.90. Understading of role of mental health comedical staff, including psychiatric social workers (PSW), and appropriate utilization.91. Appropriate utilization of communitymental health services.92. Maintainence of confidentiality of patients information93. Explanation of basic patient rights94. Assistance with decision-making in appropriate patients.95. Fromaulation of treatment plans 96. Appropriate utilization of diagnostic and management guidelines and algorithms.97. Participation in the training of younger physicians regarding clinical knowledge and skills.98. Appropriate role modelling for younger physicians.Scores were recorded on a 4-point Likert scale as follows: 1 = very confident to perform independently; 2 = fairly confident to perform independently; 3 = no confidence to perform independently; and 4 = cannot perform.1. insomnia2. edema3. lymphadenopathy4. rash5. fever6. headache7. dizziness8. vision disorder, restricted vision9. redness of the conjunctiva10. chest pain11. palpitations12. dyspnea13. cough/productive cough14. nausea/vomiting15. abnominal pain16. abnormality of bowels (diarrhea/constipation)17. back pain18. limb paralysis19. hematuria20. dysuria/incontinence21. anxiety/dysphoria22. cardiopulmonary arrest23. shock24. disturbance of consciousness25. cerebrovascular disease26. acute heart failure27. acute coronary syndromes28. acute abdomen29. acute gastrointestinal bleeding30. trauma31. acute intoxication32. aspiration33. burn injury34. suicide attempt35. anemia (iron-definciency/secondary anemia)36. cerebrovascular disease 37. eczema, dermatitis (contact/atopic)38. urticaria39. cellulitis40. bone fracture41. dislocations/sprains/tendinopathy42. osteoporosis43. spinal column disorder (disc herniation)44. cardiac arrest45. angina/myocardial infarct46. cardiac dysrhythmia (tachyarrhythmia/bradyarrhythmia)47. arterial disease (arteriosclerosis/aortic aneurysm)48. hypertension 49. respiratory failure50. respiratory tract infection (acute URI､ bronchitis､ pneumonia)51. obstructive and restrictive pulmonary disease52. diseases of upper GI tract 53. diseases of lower GI tract 54. hepatic diseases 55. disease of diaphragm, abdominal wall, and peritoneum (peritonitis/acute abdomen/hernia)56. Renal diseases(AKI/CKD/ESRD)57. disease of urological tract(urolithiasis/UTI)58. obstetrics 59. male urologic disease(prostate disease/erectile dysfunction/testicular tumor)60. metabolic disorders (DM/DM complications/hypoglycemia)61. hyperlipidemia62. ametropia(near-sighted/far-sighted/astigmatism)63. keratoconjunctivitis64. cataract65. glaucoma66. tympanitis67. allergic rhinitis68. Dementia(including secondary demantia)69. Depression70. schizophreniform disease71. somatoform disorder/stress72. viral infections (influenza/measles/rubella/chickenpox/herpes/mumps)73. bacterial infections (Staphylococcu/MRSA/group A streptococcus/chlamydia)74. tuberculosis75. rheumatic gout76. allergic disease77. pediatric seizure78. pediatric viral infections (measles､mumps､chickenpox､roseola､influenza)79. pediatric asthma80. geriatric nutritional disorders81. geriatric disease 82. death pronouncement83. post-mortem documentation84. CPC report(autopsy report)85. referral documentationScores were recorded on recorded on a 4-point Likert scale as follows: 1 = no cases, 2 = 1-5 cases, 3 = 6-10 cases, or 4 = ≥11 cases.The authors declare that they have no competing interests.Study design: SO, OT, GD, TF. Data collection: SO, OT, GD, TF and all members of the Ministry of Health, Labour and Welfare of Japan “Residency Training System and Career Path.” Statistical analysis: SO, OT, GD. First draft: SO. Critical revision of the manuscript: OT, GD, TF. Supervision and project guidance: TF. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/14/141/prepub