Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

Using Genomics to Design a Pathovar-Specific Loop-Mediated Isothermal Amplification (LAMP) Assay, for the Improved Detection of Xanthomonas citri pv. citri.

The ability to swiftly respond to pathogen incursions relies heavily on fast and accurate diagnostics. Current published assays for citrus bacterial canker do not target Xanthomonas citri pv. citri, the causative agent, with high specificity when testing Australian samples. While the current diagnostics are useful in countries where canker is endemic, the detection of canker in Australia requires an emergency response. Close relatives to X. citri pv. citri found in Australia may generate false positives with the current recommended diagnostic assays. Therefore, we developed a more specific detection tool for citrus bacterial canker to provide greater diagnostic confidence for surveillance and eradication efforts. We used genomic comparisons of 161 Xanthomonad genomes and identified and confirmed genomic regions specific for X. citri pv. citri by performing local alignments of unique regions to reference genomes. We then developed loop-mediated isothermal amplification primers and validated them against a panel of 190 isolates to confirm specificity. Our diagnostic assay showed 100% corroboration with the concurrently developed multiplex primers and represents an improved diagnostic method capable of effective citrus bacterial canker identification.

Phylogenetic diversity analysis of shotgun metagenomic reads describes gut microbiome development and treatment effects in the post-weaned pig.

Intensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. Although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life. Shotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 post-weaning piglets and 42 sows were analysed in a phylogenetic framework. Differences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment. The discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors.

Dynamics of the Queensland Fruit Fly Microbiome through the Transition from Nature to an Established Laboratory Colony.

The transition from nature to laboratory or mass rearing can impose significant physiological and evolutionary impact on insects. The Queensland fruit fly (also known as 'Qfly'), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is a serious economic pest that presents major challenges for horticulture industries in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to suppress populations in regions where this species is endemic. The biology of Qfly is intimately connected to its microbiome. Therefore, changes in the microbiome that occur through domestication have implications for SIT. There are numerous studies of the microbiome in Qfly larvae and adults, but there is little information on how the microbiome changes as Qfly laboratory colonies are established. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared from wild larvae, collected from fruit, for five generations, on a gel-based larval diet. Beta diversity analysis showed that the bacterial communities from Generation 5 (G5) clustered separately from earlier generations. At the genus level, bacterial communities were significantly different between the generations and mostly altered at G5. However, communities were found similar at phyla to family taxonomic levels. We observed high abundance of Morganella and Burkholderia at the genus level in the larval and pupal stages respectively at G5, but these were not detected in earlier generations. Overall, our findings demonstrate that the domestication process strongly affects the Qfly microbiome and prompts questions about the functional relationship between the Qfly and its microbiome, as well as implications for the performance of insects that have been domesticated and mass-reared for SIT programs.

Post-weaning shifts in microbiome composition and metabolism revealed by over 25 000 pig gut metagenome-assembled genomes.

Using a previously described metagenomics dataset of 27 billion reads, we reconstructed over 50 000 metagenome-assembled genomes (MAGs) of organisms resident in the porcine gut, 46.5 % of which were classified as >70 % complete with a <10 % contamination rate, and 24.4 % were nearly complete genomes. Here, we describe the generation and analysis of those MAGs using time-series samples. The gut microbial communities of piglets appear to follow a highly structured developmental programme in the weeks following weaning, and this development is robust to treatments including an intramuscular antibiotic treatment and two probiotic treatments. The high resolution we obtained allowed us to identify specific taxonomic 'signatures' that characterize the gut microbial development immediately after weaning. Additionally, we characterized the carbohydrate repertoire of the organisms resident in the porcine gut. We tracked the abundance shifts of 294 carbohydrate active enzymes, and identified the species and higher-level taxonomic groups carrying each of these enzymes in their MAGs. This knowledge can contribute to the design of probiotics and prebiotic interventions as a means to modify the piglet gut microbiome.

A large-scale metagenomic survey dataset of the post-weaning piglet gut lumen.

BACKGROUND: Early weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. As an alternative to antibiotic treatment, studies have previously investigated the potential of probiotics for the prevention of postweaning diarrhea. In order to describe the post-weaning gut microbiota, and to study the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we sampled and processed over 800 faecal time-series samples from 126 piglets and 42 sows. RESULTS: Here we report on the largest shotgun metagenomic dataset of the pig gut lumen microbiome to date, consisting of >8 Tbp of shotgun metagenomic sequencing data. The animal trial, the workflow from sample collection to sample processing, and the preparation of libraries for sequencing, are described in detail. We provide a preliminary analysis of the dataset, centered on a taxonomic profiling of the samples, and a 16S-based beta diversity analysis of the mothers and the piglets in the first 5 weeks after weaning. CONCLUSIONS: This study was conducted to generate a publicly available databank of the faecal metagenome of weaner piglets aged between 3 and 9 weeks old, treated with different probiotic formulations and intramuscular antibiotic treatment. Besides investigating the effects of the probiotic and intramuscular antibiotic treatment, the dataset can be explored to assess a wide range of ecological questions with regards to antimicrobial resistance, host-associated microbial and phage communities, and their dynamics during the aging of the host.

Comparative Genomics of Xanthomonas citri pv. citri A* Pathotype Reveals Three Distinct Clades with Varying Plasmid Distribution.

Citrus bacterial canker (CBC) is an important disease of citrus cultivars worldwide that causes blister-like lesions on host plants and leads to more severe symptoms such as plant defoliation and premature fruit drop. The causative agent, Xanthomonas citri pv. citri, exists as three pathotypes-A, A*, and Aw-which differ in their host range and elicited host response. To date, comparative analyses have been hampered by the lack of closed genomes for the A* pathotype. In this study, we sequenced and assembled six CBC isolates of pathotype A* using second- and third-generation sequencing technologies to produce complete, closed assemblies. Analysis of these genomes and reference A, A*, and Aw sequences revealed genetic groups within the A* pathotype. Investigation of accessory genomes revealed virulence factors, including type IV secretion systems and heavy metal resistance genes, differentiating the genetic groups. Genomic comparisons of closed genome assemblies also provided plasmid distribution information for the three genetic groups of A*. The genomes presented here complement existing closed genomes of A and Aw pathotypes that are publicly available and open opportunities to investigate the evolution of X. citri pv. citri and the virulence factors that contribute to this serious pathogen.

Artificial Larval Diet Mediates the Microbiome of Queensland Fruit Fly.

Larval diets used for artificial rearing can have a significant effect on insect biology. The Queensland fruit fly (aka "Qfly"), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is one of the greatest challenges for fruit growers in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to reduce populations in regions where this species is endemic. Factory scale rearing is essential for SIT; however, artificial larval diets are known to affect the microbiome of Qfly, which may then affect fly performance. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared, for five generations from nature, on two common artificial diets (carrot and gel). At generation five (G5), the microbiome was assessed in larvae, pupae, adult males and adult females and standard fly quality control parameters were assessed together with additional performance measures of mating propensity and survival under nutritional stress. At the genus level, bacterial communities were significantly different between the colonies reared on the two larval diets. However, communities converged at Phyla to family taxonomic levels. Bacterial genera of Morganella, Citrobacter, Providencia, and Burkholderia were highly abundant in all developmental stages of Qfly reared on the gel diet, when compared to the carrot diet. Despite abundance of these genera, a greater percentage of egg hatching, heavier pupal weight and a higher percentage of fliers were found in the Qfly reared on the gel diet. Mating propensity and survival under nutritional stress was similar for adult Qfly that had been reared on the two larval diets. Overall, our findings demonstrate that the artificial larval diet strongly influences the microbiome and quality control measures of Qfly, with likely downstream effects on performance of flies released in SIT programs.

Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.

Whole Genome Sequencing Analysis of Porcine Faecal Commensal Escherichia coli Carrying Class 1 Integrons from Sows and Their Offspring.

Intensive pig production systems often rely on the use of antimicrobials and heavy metal feed additives to maintain animal health and welfare. To gain insight into the carriage of antimicrobial resistance genes (ARGs) in the faecal flora of commercially reared healthy swine, we characterised the genome sequences of 117 porcine commensal E. coli that carried the class 1 integrase gene (intI1+). Isolates were sourced from 42 healthy sows and 126 of their offspring from a commercial breeding operation in Australia in 2017. intI1+ E. coli was detected in 28/42 (67%) sows and 90/126 (71%) piglets. Phylogroup A, particularly clonal complex 10, and phylogroup B1 featured prominently in the study collection. ST10, ST20, ST48 and ST361 were the dominant sequence types. Notably, 113/117 isolates (96%) carried three or more ARGs. Genes encoding resistance to -lactams, aminoglycosides, trimethoprim, sulphonamides, tetracyclines and heavy metals were dominant. ARGs encoding resistance to last-line agents, such as carbapenems and third generation cephalosporins, were not detected. IS26, an insertion sequence noted for its ability to capture and mobilise ARGs, was present in 108/117 (92%) intI1+ isolates, and it played a role in determining class 1 integron structure. Our data shows that healthy Australian pig faeces are an important reservoir of multidrug resistant E. coli that carry genes encoding resistance to multiple first-generation antibiotics and virulence-associated genes.

Genomic Characterisation of a Multiple Drug Resistant IncHI2 ST4 Plasmid in Escherichia coli ST744 in Australia.

Antibiotic resistance genes (ARGs) including those from the blaCTX-M family and mcr-1 that encode resistance to extended spectrum ß-lactams and colistin, respectively, have been linked with IncHI2 plasmids isolated from swine production facilities globally but not in IncHI2 plasmids from Australia. Here we describe the first complete sequence of a multiple drug resistance Australian IncHI2-ST4 plasmid, pTZ41_1P, from a commensal E. coli from a healthy piglet. pTZ41_1P carries genes conferring resistance to heavy-metals (copper, silver, tellurium and arsenic), ß-lactams, aminoglycosides and sulphonamides. The ARGs reside within a complex resistance locus (CRL) that shows considerable sequence identity to a CRL in pSDE_SvHI2, an IncHI2:ST3 plasmid from an enterotoxigenic E. coli with serotype O157:H19 of porcine origin that caused substantial losses to swine production operations in Australia in 2007. pTZ41_1P is closely related to IncHI2 plasmids found in E. coli and Salmonella enterica from porcine, avian and human sources in Europe and China but it does not carry genes encoding resistance to clinically-important antibiotics. We identified regions of IncHI2 plasmids that contribute to the genetic plasticity of this group of plasmids and highlight how they may readily acquire new resistance gene cargo. Genomic surveillance should be improved to monitor IncHI2 plasmids.

Microbiome of the Queensland Fruit Fly through Metamorphosis.

Bactroceratryoni (Froggatt) (Queensland fruit fly, or "Qfly") is a highly polyphagous tephritid fruit fly and a serious economic pest in Australia. Qfly biology is intimately linked to the bacteria and fungi of its microbiome. While there are numerous studies of the microbiome in larvae and adults, the transition of the microbiome through the pupal stage remains unknown. To address this knowledge gap, we used high-throughput Next-Generation Sequencing (NGS) to examine microbial communities at each developmental stage in the Qfly life cycle, targeting the bacterial 16S rRNA and fungal ITS regions. We found that microbial communities were similar at the larval and pupal stage and were also similar between adult males and females, yet there were marked differences between the larval and adult stages. Specific bacterial and fungal taxa are present in the larvae and adults (fed hydrolyzed yeast with sugar) which is likely related to differences in nutritional biology of these life stages. We observed a significant abundance of the Acetobacteraceae at the family level, both in the larval and pupal stages. Conversely, Enterobacteriaceae was highly abundant (> 80%) only in the adults. The majority of fungal taxa present in Qfly were yeasts or yeast-like fungi. In addition to elucidating changes in the microbiome through developmental stages, this study characterizes the Qfly microbiome present at the establishment of laboratory colonies as they enter the domestication process.

Genomic Acquisitions in Emerging Populations of Xanthomonas vasicola pv. vasculorum Infecting Corn in the United States and Argentina.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

Commensal microbiota modulates larval foraging behaviour, development rate and pupal production in Bactrocera tryoni.

BACKROUND: Commensal microbes can promote survival and growth of developing insects, and have important fitness implications in adulthood. Insect larvae can acquire commensal microbes through two main routes: by vertical acquisition from maternal deposition of microbes on the eggshells and by horizontal acquisition from the environment where the larvae develop. To date, however, little is known about how microbes acquired through these different routes interact to shape insect development. In the present study, we investigated how vertically and horizontally acquired microbiota influence larval foraging behaviour, development time to pupation and pupal production in the Queensland fruit fly ('Qfly'), Bactrocera tryoni. RESULTS: Both vertically and horizontally acquired microbiota were required to maximise pupal production in Qfly. Moreover, larvae exposed to both vertically and horizontally acquired microbiota pupated sooner than those exposed to no microbiota, or only to horizontally acquired microbiota. Larval foraging behaviour was also influenced by both vertically and horizontally acquired microbiota. Larvae from treatments exposed to neither vertically nor horizontally acquired microbiota spent more time overall on foraging patches than did larvae of other treatments, and most notably had greater preference for diets with extreme protein or sugar compositions. CONCLUSION: The integrity of the microbiota early in life is important for larval foraging behaviour, development time to pupation, and pupal production in Qflies. These findings highlight the complexity of microbial relations in this species, and provide insights to the importance of exposure to microbial communities during laboratory- or mass-rearing of tephritid fruit flies.

Diet and irradiation effects on the bacterial community composition and structure in the gut of domesticated teneral and mature Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

BACKGROUND: Mass-rearing, domestication and gamma irradiation of tephritid fruit flies used in sterile insect technique (SIT) programmes can negatively impact fly quality and performance. Symbiotic bacteria supplied as probiotics to mass-reared fruit flies may help to overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known. RESULTS: We have used next-generation sequencing to characterise the bacterial community composition and structure within Queensland fruit fly, Bactrocera tryoni (Froggatt), by generating 16S rRNA gene amplicon libraries derived from the guts of 58 individual teneral and mature, female and male, sterile and fertile adult flies reared on artificial larval diets in a laboratory or mass-rearing environment, and fed either a full adult diet (i.e. sugar and yeast hydrolysate) or a sugar only adult diet. Overall, the amplicon sequence read volume in tenerals was low and smaller than in mature adult flies. Operational taxonomic units (OTUs), belonging to the families Enterobacteriaceae (8 OTUs) and Acetobacteraceae (1 OTU) were most prevalent. Enterobacteriaceae dominated laboratory-reared tenerals from a colony fed a carrot-based larval diet, while Acetobacteraceae dominated mass-reared tenerals from a production facility colony fed a lucerne chaff based larval diet. As adult flies matured, Enterobacteriaceae became dominant irrespective of larval origin. The inclusion of yeast in the adult diet strengthened this shift away from Acetobacteraceae towards Enterobacteriaceae. Interestingly, irradiation increased 16S rRNA gene sequence read volume. CONCLUSIONS: Our findings suggest that bacterial populations in fruit flies experience significant bottlenecks during metamorphosis. Gut bacteria in teneral flies were less abundant and less diverse, and impacted by colony origin. In contrast, mature adult flies had selectively increased abundances for some gut bacteria, or acquired these bacteria from the adult diet and environment. Furthermore, irradiation augmented bacterial abundance in mature flies. This implies that either some gut bacteria were compensating for damage caused by irradiation or irradiated flies had lost their ability to regulate bacterial load. Our findings suggest that the adult stage prior to sexual maturity may be ideal to target for probiotic manipulation of fly microbiota to increase fly performance in SIT programmes.

Tephritid-microbial interactions to enhance fruit fly performance in sterile insect technique programs.

BACKGROUND: The Sterile Insect Technique (SIT) is being applied for the management of economically important pest fruit flies (Diptera: Tephritidae) in a number of countries worldwide. The success and cost effectiveness of SIT depends upon the ability of mass-reared sterilized male insects to successfully copulate with conspecific wild fertile females when released in the field. METHODS: We conducted a critical analysis of the literature about the tephritid gut microbiome including the advancement of methods for the identification and characterization of microbiota, particularly next generation sequencing, the impacts of irradiation (to induce sterility of flies) and fruit fly rearing, and the use of probiotics to manipulate the fruit fly gut microbiota. RESULTS: Domestication, mass-rearing, irradiation and handling, as required in SIT, may change the structure of the fruit flies' gut microbial community compared to that of wild flies under field conditions. Gut microbiota of tephritids are important in their hosts' development, performance and physiology. Knowledge of how mass-rearing and associated changes of the microbial community impact the functional role of the bacteria and host biology is limited. Probiotics offer potential to encourage a gut microbial community that limits pathogens, and improves the quality of fruit flies. CONCLUSIONS: Advances in technologies used to identify and characterize the gut microbiota will continue to expand our understanding of tephritid gut microbial diversity and community composition. Knowledge about the functions of gut microbes will increase through the use of gnotobiotic models, genome sequencing, metagenomics, metatranscriptomics, metabolomics and metaproteomics. The use of probiotics, or manipulation of the gut microbiota, offers significant opportunities to enhance the production of high quality, performing fruit flies in operational SIT programs.

Next-Generation Sequencing reveals relationship between the larval microbiome and food substrate in the polyphagous Queensland fruit fly.

Insects typically host substantial microbial communities (the 'microbiome') that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni ('Queensland fruit fly') that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect's microbiome and that of the surrounding host tissues.

Escherichia coli ST302: Genomic Analysis of Virulence Potential and Antimicrobial Resistance Mediated by Mobile Genetic Elements.

aEPEC are associated with persistent diarrhea, and diarrheal outbreaks in both humans and animals worldwide. They are differentiated from typical EPEC by the lack of bundle-forming pili, and from EHEC by the lack of phage-mediated stx toxins. However, phylogenetic analyses often associate aEPEC with EHEC, promoting the hypothesis that aEPEC are the progenitors of EHEC, which is supported by aEPEC conversion to EHEC by stx-carrying phages. While aEPEC can cause disease outright, the potential to acquire stx, one of the most potent bacterial toxins known, merits close monitoring. Escherichia coli ST302 (O108:H9, O182:H9, O45:H9) are aEPEC that have been isolated from diarrheic human, pig and rabbit hosts, as well as in healthy pigs, however, no study to date has focused on E. coli ST302 strains. Through WGS and hybrid assembly we present the first closed chromosome, and two circularized plasmids of an ST302 strain - F2_18C, isolated from a healthy pig in Australia. A phylogenetic analysis placed E. coli ST302 strains in proximity to EHEC ST32 (O145:H28) strains. Public databases were interrogated for WGSs of E. coli ST302 strains and short-read gene screens were used to compare their virulence-associated gene (VAG) and antimicrobial resistance gene (ARG) cargo. E. coli ST302 strains carry diverse VAGs, including those that typically associated with extraintestinal pathogenic E. coli (ExPEC). Plasmid comparisons showed that pF2_18C_FIB shared homology with EHEC virulence plasmids such as pO103 while pF2_18C_HI2 is a large multidrug resistance IncHI2:ST3 plasmid. A comparison of 33 HI2:ST3 plasmids demonstrated that those of Australian origin have not acquired resistances to extended-spectrum beta-lactams, colistin, fosfomycin or rifampicin, unlike those originating from Asia. F2_18C was shown to carry two additional pathogenicity islands - ETT2, and the STEC-associated PAI CL 3, plasmid-associated heavy metal resistance genes, as well as several unoccupied stx-phage attachment sites. This study sheds light on the virulence and AMR potential of E. coli ST302 strains and informs AMR genomic surveillance.