Comparative Genomics of <i>Xanthomonas citri</i> pv. <i>citri</i> 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

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 β-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), β-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

Near full-length 16S rRNA gene next-generation sequencing revealed Asaia as a common midgut bacterium of wild and domesticated Queensland fruit fly larvae

BACKGROUND: Gut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (> 1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets). RESULTS: Near full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains. CONCLUSIONS: Variation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT

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

Complete Sequences of Multiple-Drug Resistant IncHI2 ST3 Plasmids in Escherichia coli of Porcine Origin in Australia

© Copyright © 2019 Wyrsch, Reid, DeMaere, Liu, Chapman, Roy Chowdhury and Djordjevic. IncHI2 ST3 plasmids are known carriers of multiple antimicrobial resistance genes. Complete plasmid sequences from multiple drug resistant Escherichia coli circulating in Australian swine is however limited. Here we sequenced two related IncHI2 ST3 plasmids, pSDE-SvHI2, and pSDC-F2_12BHI2, from phylogenetically unrelated multiple-drug resistant Escherichia coli strains SvETEC (CC23:O157:H19) and F2_12B (ST93:O7:H4) from geographically disparate pig production operations in New South Wales, Australia. Unicycler was used to co-assemble short read (Illumina) and long read (PacBio SMRT) nucleotide sequence data. The plasmids encoded three drug-resistance loci, two of which carried class 1 integrons. One integron, hosting drfA12-orfF-aadA2, was within a hybrid Tn1721/Tn21, with the second residing within a copper/silver resistance transposon, comprising part of an atypical sul3-associated structure. The third resistance locus was flanked by IS15DI and encoded neomycin resistance (neoR). An oqx-encoding transposon (quinolone resistance), similar in structure to Tn6010, was identified only in pSDC-F2_12BHI2. Both plasmids showed high sequence identity to plasmid pSTM6-275, recently described in Salmonella enterica serotype 1,4,[5],12:i:- that has risen to prominence and become endemic in Australia. IncHI2 ST3 plasmids circulating in commensal and pathogenic E. coli from Australian swine belong to a lineage of plasmids often in association with sul3 and host multiple complex antibiotic and metal resistance structures, formed in part by IS26

Porcine commensal escherichia coli: A reservoir for class 1 integrons associated with IS26

© 2017 The Authors. Porcine faecal waste is a serious environmental pollutant. Carriage of antimicrobial-resistance genes (ARGs) and virulenceassociated genes (VAGs), and the zoonotic potential of commensal Escherichia coli from swine are largely unknown. Furthermore, little is known about the role of commensal E. coli as contributors to the mobilization of ARGs between food animals and the environment. Here, we report whole-genome sequence analysis of 103 class 1 integron-positive E. coli from the faeces of healthy pigs from two commercial production facilities in New South Wales, Australia. Most strains belonged to phylogroups A and B1, and carried VAGs linked with extraintestinal infection in humans. The 103 strains belonged to 37 multilocus sequence types and clonal complex 10 featured prominently. Seventeen ARGs were detected and 97% (100/103) of strains carried three or more ARGs. Heavy-metal-resistance genes merA, cusA and terA were also common. IS26 was observed in 98% (101/103) of strains and was often physically associated with structurally diverse class 1 integrons that carried unique genetic features, which may be tracked. This study provides, to our knowledge, the first detailed genomic analysis and point of reference for commensal E. coli of porcine origin in Australia, facilitating tracking of specific lineages and the mobile resistance genes they carry

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

Comparative genomic analysis of a multiple antimicrobial resistant enterotoxigenic E. coli O157 lineage from Australian pigs

© 2015 Wyrsch et al. Background: Enterotoxigenic Escherichia coli (ETEC) are a major economic threat to pig production globally, with serogroups O8, O9, O45, O101, O138, O139, O141, O149 and O157 implicated as the leading diarrhoeal pathogens affecting pigs below four weeks of age. A multiple antimicrobial resistant ETEC O157 (O157 SvETEC) representative of O157 isolates from a pig farm in New South Wales, Australia that experienced repeated bouts of pre- and post-weaning diarrhoea resulting in multiple fatalities was characterized here. Enterohaemorrhagic E. coli (EHEC) O157:H7 cause both sporadic and widespread outbreaks of foodborne disease, predominantly have a ruminant origin and belong to the ST11 clonal complex. Here, for the first time, we conducted comparative genomic analyses of two epidemiologically-unrelated porcine, disease-causing ETEC O157; E. coli O157 SvETEC and E. coli O157:K88 734/3, and examined their phylogenetic relationship with EHEC O157:H7. Results: O157 SvETEC and O157:K88 734/3 belong to a novel sequence type (ST4245) that comprises part of the ST23 complex and are genetically distinct from EHEC O157. Comparative phylogenetic analysis using PhyloSift shows that E. coli O157 SvETEC and E. coli O157:K88 734/3 group into a single clade and are most similar to the extraintestinal avian pathogenic Escherichia coli (APEC) isolate O78 that clusters within the ST23 complex. Genome content was highly similar between E. coli O157 SvETEC, O157:K88 734/3 and APEC O78, with variability predominantly limited to laterally acquired elements, including prophages, plasmids and antimicrobial resistance gene loci. Putative ETEC virulence factors, including the toxins STb and LT and the K88 (F4) adhesin, were conserved between O157 SvETEC and O157:K88 734/3. The O157 SvETEC isolate also encoded the heat stable enterotoxin STa and a second allele of STb, whilst a prophage within O157:K88 734/3 encoded the serum survival gene bor. Both isolates harbor a large repertoire of antibiotic resistance genes but their association with mobile elements remains undetermined. Conclusions: We present an analysis of the first draft genome sequences of two epidemiologically-unrelated, pathogenic ETEC O157. E. coli O157 SvETEC and E. coli O157:K88 734/3 belong to the ST23 complex and are phylogenetically distinct to EHEC O157 lineages that reside within the ST11 complex

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

BackgroundEarly 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.ResultsHere 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.ConclusionsThis 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

The Verticillium wilt problem in Australian cotton

© 2021, Australasian Plant Pathology Society Inc. Verticillium dahliae is a soil-borne phytopathogen and the causal agent of Verticillium wilt. It affects many agriculturally important crops around the world, including cotton. In Australia, the billion-dollar cotton industry is increasingly impacted by Verticillium wilt. Internationally it has been reported that the defoliating V. dahliae Vegetative Compatibility Group (VCG) 1A causes severe damage to cotton. In Australia however, the non-defoliating VCG2A is causing more severe damage to crops in fields than the defoliating VCG1A. This review examines the current research to understand the Australian V. dahliae situation, including current classification systems, genetic analyses and management strategies. It appears that virulence cannot be defined solely by VCG in Australian Verticillium dahliae isolates causing disease in cotton, and that the industry must continually adapt their practices in order to keep the disease under control