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Globally, the anaerobic bacterium Clostridium perfringens causes severe disease in a wide array of hosts; however, C. perfringens strains are also carried asymptomatically. Accessory genes are responsible for much of the observed phenotypic variation and virulence within this species, with toxins frequently encoded on conjugative plasmids and many isolates carrying up to 10 plasmids. Despite this unusual biology, current genomic analyses have largely excluded isolates from healthy hosts or environmental sources. Accessory genomes, including plasmids, also have often been excluded from broader scale phylogenetic investigations. Here we interrogate a comprehensive collection of 464 C. perfringens genomes and identify the first putative non-conjugative enterotoxin (CPE)-encoding plasmids and a putative novel conjugative locus (Bcp) with sequence similarity to a locus reported from Clostridium botulinum. We sequenced and archived 102 new C. perfringens genomes, including those from rarely sequenced toxinotype B, C, D and E isolates. Long-read sequencing of 11 C. perfringens strains representing all toxinotypes (A-G) identified 55 plasmids from nine distinct plasmid groups. Interrogation of the 464 genomes in this collection identified 1045 plasmid-like contigs from the nine plasmid families, with a wide distribution across the C. perfringens isolates. Plasmids and plasmid diversity play an essential role in C. perfringens pathogenicity and broader biology. We have expanded the C. perfringens genome collection to include temporal, spatial and phenotypically diverse isolates including those carried asymptomatically in the gastrointestinal microbiome. This analysis has resulted in the identification of novel C. perfringens plasmids whilst providing a comprehensive understanding of species diversity.

BACKGROUND: Health care and well-being are 2 main interconnected application areas of conversational agents (CAs). There is a significant increase in research, development, and commercial implementations in this area. In parallel to the increasing interest, new challenges in designing and evaluating CAs have emerged. OBJECTIVE: This study aims to identify key design, development, and evaluation challenges of CAs in health care and well-being research. The focus is on the very recent projects with their emerging challenges. METHODS: A review study was conducted with 17 invited studies, most of which were presented at the ACM (Association for Computing Machinery) CHI 2020 conference workshop on CAs for health and well-being. Eligibility criteria required the studies to involve a CA applied to a health or well-being project (ongoing or recently finished). The participating studies were asked to report on their projects' design and evaluation challenges. We used thematic analysis to review the studies. RESULTS: The findings include a range of topics from primary care to caring for older adults to health coaching. We identified 4 major themes: (1) Domain Information and Integration, (2) User-System Interaction and Partnership, (3) Evaluation, and (4) Conversational Competence. CONCLUSIONS: CAs proved their worth during the pandemic as health screening tools, and are expected to stay to further support various health care domains, especially personal health care. Growth in investment in CAs also shows the value as a personal assistant. Our study shows that while some challenges are shared with other CA application areas, safety and privacy remain the major challenges in the health care and well-being domains. An increased level of collaboration across different institutions and entities may be a promising direction to address some of the major challenges that otherwise would be too complex to be addressed by the projects with their limited scope and budget.

Ulcerative colitis is an inflammatory disease of the colon that is associated with colonic neutrophil accumulation. Recent evidence indicates that diet alters the composition of the gut microbiota and influences host-pathogen interactions. Specifically, bacterial fermentation of dietary fiber produces metabolites called short-chain fatty acids (SCFAs), which have been shown to protect against various inflammatory diseases. However, the effect of fiber deficiency on the key initial steps of inflammation, such as leukocyte-endothelial cell interactions, is unknown. Moreover, the impact of fiber deficiency on neutrophil recruitment under basal conditions and during inflammation in vivo is unknown. Herein, we hypothesized that a fiber-deficient diet promotes an inflammatory state in the colon at baseline and predisposes the host to more severe colitis pathology. Mice fed a no-fiber diet for 14 days showed significant changes in the gut microbiota and exhibited increased neutrophil-endothelial interactions in the colonic microvasculature. Although mice fed a no-fiber diet alone did not have observable colitis-associated symptoms, these animals were highly susceptible to low dose (0.5%) dextran sodium sulphate (DSS)-induced model of colitis. Supplementation of the most abundant SCFA, acetate, prevented no-fiber diet-mediated enrichment of colonic neutrophils and colitis pathology. Therefore, dietary fiber, possibly through the actions of acetate, plays an important role in regulating neutrophil recruitment and host protection against inflammatory colonic damage in an experimental model of colitis.

The microbiota is considered critical for normal vertebrate homeostasis and it may exert its effects at a local level within the gastrointestinal tract, or systemically through the production of bacterial metabolites. To date, investigations into the role that the microbiota plays in reptile physiology are rare. To address this knowledge gap, we explored the relationship between differences in microbial communities to see if they accounted for differences in haematology and biochemistry values, in different populations of nesting flatback turtles (Natator depressus). We found that microbiota composition was not correlated to any of the blood analytes we measured in flatbacks. This study is the first of its kind in reptiles and highlights the need for further investigations to determine mechanisms by which the microbiota influences the physiology and health of reptiles.

BACKGROUND: The microbiota plays a critical role in host homeostasis and has been shown to be a major driving force in host evolution. However, our understanding of these important relationships is hampered by a lack of data for many species, and by significant gaps in sampling of the evolutionary tree. In this investigation we improve our understanding of the host-microbiome relationship by obtaining samples from all seven extant species of sea turtle, and correlate microbial compositions with host evolutionary history. RESULTS: Our analysis shows that the predominate phyla in the microbiota of nesting sea turtles was Proteobacteria. We also demonstrate a strong relationship between the bacterial phyla SR1 and sea turtle phylogeny, and that sea turtle microbiotas have changed very slowly over time in accordance with their similarly slow phenotypic changes. CONCLUSIONS: This is one of the most comprehensive microbiota studies to have been performed in a single clade of animals and further improves our knowledge of how microbial populations have influenced vertebrate evolution.

Conversational agents have increasingly been deployed in healthcare applications. However, significant challenges remain in developing this technology. Recent research in this area has highlighted that: i) patient safety was rarely evaluated; ii) health outcomes were poorly measured, and iii) no standardised evaluation methods were employed. The conversational agents in healthcare are lagging behind the developments in other domains. This one-day workshop aims to create a roadmap for healthcare conversational agents to develop standardised design and evaluation frameworks. This will prioritise health outcomes and patient safety while ensuring a high-quality user experience. In doing so, this workshop will bring together researchers and practitioners from HCI, healthcare and related speech and chatbot domains to collaborate on these key challenges.

Clostridium perfringens causes a wide range of diseases in a variety of hosts, due to the production of a diverse set of toxins and extracellular enzymes. The C. perfringens toxins play an important role in pathogenesis, such that the presence and absence of the toxins is used as a typing scheme for the species. In recent years, several new toxins have been discovered that have been shown to be essential or highly correlated to diseases; these include binary enterotoxin (BecAB), NetB and NetF. In the current study, genome sequence analysis of C. perfringens isolates from diverse sources revealed several putative novel toxin homologs, some of which appeared to be associated with potential mobile genetic elements, including transposons and plasmids. Four novel toxin homologs encoding proteins related to the pore-forming Leukocidin/Hemolysin family were found in type A and G isolates. Two novel toxin homologs encoding proteins related to the epsilon aerolysin-like toxin family were identified in Type A and F isolates from humans, contaminated food and turkeys. A novel set of proteins related to clostridial binary toxins was also identified. While phenotypic characterisation is required before any of these homologs can be established as functional toxins, the in silico identification of these novel homologs on mobile genetic elements suggests the potential toxin reservoir of C. perfringens may be much larger than previously thought.

Chickens infected with Campylobacter jejuni or Campylobacter coli are largely asymptomatic, however, infection with the closely related species, Campylobacter hepaticus, can result in Spotty Liver Disease (SLD). C. hepaticus has been detected in the liver, bile, small intestine and caecum of SLD affected chickens. The survival and colonization mechanisms that C. hepaticus uses to colonize chickens remain unknown. In this study, we compared the genome sequences of 14 newly sequenced Australian isolates of C. hepaticus, isolates from outbreaks in the United Kingdom, and reference strains of C. jejuni and C. coli, with the aim of identifying virulence genes associated with SLD. We also carried out global comparative transcriptomic analysis between C. hepaticus recovered from the bile of SLD infected chickens and C. hepaticus grown in vitro. This revealed how the bacteria adapt to proliferate in the challenging host environment in which they are found. Additionally, biochemical experiments confirmed some in silico metabolic predictions. We found that, unlike other Campylobacter sp., C. hepaticus encodes glucose and polyhydroxybutyrate metabolism pathways. This study demonstrated the metabolic plasticity of C. hepaticus, which may contribute to survival in the competitive, nutrient and energy-limited environment of the chicken. Transcriptomic analysis indicated that gene clusters associated with glucose utilization, stress response, hydrogen metabolism, and sialic acid modification may play an important role in the pathogenicity of C. hepaticus. An understanding of the survival and virulence mechanisms that C. hepaticus uses will help to direct the development of effective intervention methods to protect birds from the debilitating effects of SLD.

BACKGROUND: Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. RESULTS: Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. CONCLUSIONS: The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. Th

Organophosphate insecticides have been directly or indirectly implicated in avian populations declining worldwide. Birds in agricultural environments are commonly exposed to these insecticides, mainly through ingestion of invertebrates after insecticide application. Despite insecticide exposure in birds occurring mostly by ingestion, the impact of organophosphates on the avian digestive system has been poorly researched. In this work we used the Japanese quail (Coturnix japonica) as an avian model to study short-term microbial community responses to a single dose of trichlorfon at low concentration in three sample origins of the gastrointestinal tract (GIT): caecum, large intestine and faeces. Using next-generation sequencing of 16S rRNA gene amplicons as bacterial markers, the study showed that ingestion of insecticide caused significant changes in the GIT microbiome. Specifically, microbiota composition and diversity differed between treated and untreated quail. Insecticide-associated responses in the caecum showed differences between sexes which did not occur with the other sample types. In caecal microbiota, only treated females showed significant shifts in a number of genera within the Lachnospiraceae and the Enterobacteriaceae families. The major responses in the large intestine were a significant reduction in the genus Lactobacillus and increases in abundance of a number of Proteobacteria genera. All microbial shifts in faeces occurred in phylotypes that were represented at low relative abundances. In general, changes in microbiota possibly resulted from contrasting responses towards the insecticide, either positive (e.g., biodegrading bacteria) or negative (e.g., insecticide-susceptible bacteria). This study demonstrates the significant impact that organophosphate insecticides have on the avian gut microbiota; showing that a single small dose of trichlorfon caused dysbiosis in the GIT of the Japanese quail. Further research is necessary to understand the imp

BACKGROUND: The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. RESULTS: We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. CONCLUSIONS: Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

BACKGROUND: In this report we have explored the genomic and microbiological basis for a sustained increase in bloodstream infections at a major Australian hospital caused by Enterococcus faecium multi-locus sequence type (ST) 203, an outbreak strain that has largely replaced a predecessor ST17 sequence type. RESULTS: To establish a ST203 reference sequence we fully assembled and annotated the genome of Aus0085, a 2009 vancomycin-resistant Enterococcus faecium (VREfm) bloodstream isolate, and the first example of a completed ST203 genome. Aus0085 has a 3.2 Mb genome, comprising a 2.9 Mb circular chromosome and six circular plasmids (2 kb-130 kb). Twelve percent of the 3222 coding sequences (CDS) in Aus0085 are not present in ST17 E. faecium Aus0004 and ST18 E. faecium TX16. Extending this comparison to an additional 12 ST17 and 14 ST203 E. faecium hospital isolate genomes revealed only six genomic regions spanning 41 kb that were present in all ST203 and absent from all ST17 genomes. The 40 CDS have predicted functions that include ion transport, riboflavin metabolism and two phosphotransferase systems. Comparison of the vancomycin resistance-conferring Tn1549 transposon between Aus0004 and Aus0085 revealed differences in transposon length and insertion site, and van locus sequence variation that correlated with a higher vancomycin MIC in Aus0085. Additional phenotype comparisons between ST17 and ST203 isolates showed that while there were no differences in biofilm-formation and killing of Galleria mellonella, ST203 isolates grew significantly faster and out-competed ST17 isolates in growth assays. CONCLUSIONS: Here we have fully assembled and annotated the first ST203 genome, and then characterized the genomic differences between ST17 and ST203 E. faecium. We also show that ST203 E. faecium are faster growing and can out-compete ST17 E. faecium. While a causal genetic basis for these phenotype differences is not provided here, this study revealed conserved genetic d

UNLABELLED: The pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netB was insertionally inactivated by the chloramphenicol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netB gene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained the netB gene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2 gene, which encodes a different pore-forming toxin, beta2 toxin. IMPORTANCE: The anaerobic bacterium Clostridium perfringens can cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries the netB gene is conjugative and has a 40-kb region that is very similar

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.