Life at the borderlands: microbiomes of interfaces critical to One Health.

Microbiomes are foundational components of the environment that provide essential services relating to food security, carbon sequestration, human health, and the overall well-being of ecosystems. Microbiota exert their effects primarily through complex interactions at interfaces with their plant, animal, and human hosts, as well as within the soil environment. This review aims to explore the ecological, evolutionary, and molecular processes governing the establishment and function of microbiome-host relationships, specifically at interfaces critical to One Health-a transdisciplinary framework that recognizes that the health outcomes of people, animals, plants, and the environment are tightly interconnected. Within the context of One Health, the core principles underpinning microbiome assembly will be discussed in detail, including biofilm formation, microbial recruitment strategies, mechanisms of microbial attachment, community succession, and the effect these processes have on host function and health. Finally, this review will catalogue recent advances in microbiology and microbial ecology methods that can be used to profile microbial interfaces, with particular attention to multi-omic, advanced imaging, and modelling approaches. These technologies are essential for delineating the general and specific principles governing microbiome assembly and functions, mapping microbial interconnectivity across varying spatial and temporal scales, and for the establishment of predictive frameworks that will guide the development of targeted microbiome-interventions to deliver One Health outcomes.

Genomic surveillance for antimicrobial resistance - a One Health perspective.

Antimicrobial resistance (AMR) - the ability of microorganisms to adapt and survive under diverse chemical selection pressures - is influenced by complex interactions between humans, companion and food-producing animals, wildlife, insects and the environment. To understand and manage the threat posed to health (human, animal, plant and environmental) and security (food and water security and biosecurity), a multifaceted 'One Health' approach to AMR surveillance is required. Genomic technologies have enabled monitoring of the mobilization, persistence and abundance of AMR genes and mutations within and between microbial populations. Their adoption has also allowed source-tracing of AMR pathogens and modelling of AMR evolution and transmission. Here, we highlight recent advances in genomic AMR surveillance and the relative strengths of different technologies for AMR surveillance and research. We showcase recent insights derived from One Health genomic surveillance and consider the challenges to broader adoption both in developed and in lower- and middle-income countries.

Identification of genes influencing the evolution of Escherichia coli ST372 in dogs and humans.

ST372 are widely reported as the major Escherichia coli sequence type in dogs globally. They are also a sporadic cause of extraintestinal infections in humans. Despite this, it is unknown whether ST372 strains from dogs and humans represent shared or distinct populations. Furthermore, little is known about genomic traits that might explain the prominence of ST372 in dogs or presence in humans. To address this, we applied a variety of bioinformatics analyses to a global collection of 407 ST372 E. coli whole-genome sequences to characterize their epidemiological features, population structure and associated accessory genomes. We confirm that dogs are the dominant host of ST372 and that clusters within the population structure exhibit distinctive O:H types. One phylogenetic cluster, 'cluster M', comprised almost half of the sequences and showed the divergence of two human-restricted clades that carried different O:H types to the remainder of the cluster. We also present evidence supporting transmission between dogs and humans within different clusters of the phylogeny, including M. We show that multiple acquisitions of the pdu propanediol utilization operon have occurred in clusters dominated by isolates of canine source, possibly linked to diet, whereas loss of the pdu operon and acquisition of K antigen virulence genes characterize human-restricted lineages.

Genomic and Temporal Trends in Canine ExPEC Reflect Those of Human ExPEC.

Companion animals and humans are known to share extraintestinal pathogenic Escherichia coli (ExPEC), but the extent of E. coli sequence types (STs) that cause extraintestinal diseases in dogs is not well understood. Here, we generated whole-genome sequences of 377 ExPEC collected by the University of Melbourne Veterinary Hospital from dogs over an 11-year period from 2007 to 2017. Isolates were predominantly from urogenital tract infections (219, 58.1%), but isolates from gastrointestinal specimens (51, 13.5%), general infections (72, 19.1%), and soft tissue infections (34, 9%) were also represented. A diverse collection of 53 STs were identified, with 18 of these including at least five sequences. The five most prevalent STs were ST372 (69, 18.3%), ST73 (31, 8.2%), ST127 (22, 5.8%), ST80 (19, 5.0%), and ST58 (14, 3.7%). Apart from ST372, all of these are prominent human ExPEC STs. Other common ExPEC STs identified included ST12, ST131, ST95, ST141, ST963, ST1193, ST88, and ST38. Virulence gene profiles, antimicrobial resistance carriage, and trends in plasmid carriage for specific STs were generally reflective of those seen in humans. Many of the prominent STs were observed repetitively over an 11-year time span, indicating their persistence in the dogs in the community, which is most likely driven by household sharing of E. coli between humans and their pets. The case of ST372 as a dominant canine lineage observed sporadically in humans is flagged for further investigation. IMPORTANCE Pathogenic E. coli that causes extraintestinal infections (ExPEC) in humans and canines represents a significant burden in hospital and veterinary settings. Despite the obvious interrelationship between dogs and humans favoring both zoonotic and anthropozoonotic infections, whole-genome sequencing projects examining large numbers of canine-origin ExPEC are lacking. In support of anthropozoonosis, we found that most STs from canine infections are dominant human ExPEC STs (e.g., ST73, ST127, ST131) with similar genomic traits, such as plasmid carriage and virulence gene burden. In contrast, we identified ST372 as the dominant canine ST and a sporadic cause of infection in humans, supporting zoonotic transfer. Furthermore, we highlight that, as is the case in humans, STs in canine disease are consistent over time, implicating the gastrointestinal tract as the major community reservoir, which is likely augmented by exposure to human E. coli via shared diet and proximity.

Close genetic linkage between human and companion animal extraintestinal pathogenic Escherichia coli ST127.

Escherichia coli ST127, a recently emerged global pathogen noted for high virulence gene carriage, is a leading cause of urinary tract and blood stream infections. ST127 is frequently isolated from humans and companion animals; however, it is unclear if they are distinct or related populations of ST127. We performed a phylogenomic analysis of 299 E. coli ST127 of diverse epidemiological origin to characterize their population structure, genetic determinants of virulence, antimicrobial resistance, and repertoire of mobile genetic elements with a focus on plasmids. The core gene phylogeny was divided into 13 clusters, the largest of which (BAP4) contained the majority of human and companion animal origin isolates. This dominant cluster displayed genetic differences to the remainder of the phylogeny, most notably alternative gene alleles encoding important virulence factors including lipid A, flagella, and K capsule. Furthermore, numerous close genetic linkages (<30 SNPs) between human and companion animal isolates were observed within the cluster. Carriage of antimicrobial resistance genes in the collection was limited, but virulence gene carriage was extensive. We found evidence of pUTI89-like virulence plasmid carriage in over a third of isolates, localised to four of the major phylogenetic clusters. Our study supports global scale repetitive transfer of E. coli ST127 lineages between humans and companion animals, particularly within the dominant BAP4 cluster.

A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle.

F Plasmid Lineages in Escherichia coli ST95: Implications for Host Range, Antibiotic Resistance, and Zoonoses.

Escherichia coli sequence type 95 (ST95) is an extraintestinal pathogenic E. coli (ExPEC) renowned for its ability to cause significant morbidity and mortality in humans and poultry. A core genome analysis of 668 ST95 isolates generated 10 clades (A to J), 5 of which are reported here for the first time. F plasmid replicon sequence typing showed that almost a third (178/668 [27%]) of the collection carry pUTI89 (F29:B10) and were restricted to clade A and a sublineage of clade B. In contrast, almost half (328/668 [49%]) of the collection across multiple clades harbor ColV plasmids (multiple F types). Strikingly, ST95 lineages with pUTI89 were almost exclusively from humans, while ColV+ ST95 lineages were sourced from poultry and humans. Clade I was notable because it comprises temporally and geographically matched ColV+ isolates sourced from human and retail poultry meat, suggesting interspecies transmission via food. Clade F contained ST95 isolates of bovine origin, none of which carried ColV or pUTI89 plasmids. Remarkably, an analysis of a cohort of 34,176 E. coli isolates comprising 2,570 sequence types mirrored what was observed in ST95: (i) pUTI89 was overwhelmingly linked to E. coli sourced from humans but almost entirely absent from 13,027 E. coli isolates recovered from poultry, pigs, and cattle, and (ii) E. coli isolates harboring ColV plasmids were from multiple sources, including humans, poultry, and swine. Overall, our data suggest that F plasmids influence E. coli host range, clade structure, and zoonotic potential in ST95 and ExPEC more broadly. IMPORTANCE E. coli ST95 is one of five dominant ExPEC lineages globally and noted for causing urinary tract and bloodstream infections and neonatal meningitis in humans and colibacillosis in poultry. Using high-resolution phylogenomics, we show that F replicon sequence type is linked to ST95 clade structure and zoonotic potential. Specifically, human centric ST95 clades overwhelmingly harbor F29:B10 (pUTI89) plasmids, while clades carrying both human- and poultry-sourced isolates are typically ColV+ with multiple replicon types. Importantly, several clades identified clonal ColV+ ST95 isolates from human and poultry sources, but clade I, which housed temporally and spatially matched isolates, provided the most robust evidence. Notably, patterns of association of F replicon types with E. coli host were mirrored within a diverse collection of 34,176 E. coli genomes. Our studies indicate that the role of food animals as a source of human ExPEC disease is complex and warrants further investigation.

OPA-driven hollow-core fiber as a tunable, broadband source for coherent multidimensional spectroscopy.

Despite the impressive abilities of coherent multi-dimensional spectroscopy (CMDS), its' implementation is limited due to the complexity of continuum generation and required phase stability between the pump pulse pair. In light of this, we have implemented a system producing sub-10 fs pulses with tunable central wavelength. Using a commercial OPA to drive a hollow-core fiber, the system is extremely simple. Output pulse energies lie in the 40-80 µJ range, more than sufficient for transmission through the pulse shaping optics and beam splitters necessary for CMDS. Power fluctuations are minimal, mode quality is excellent, and spectral phase is well behaved at the output. To demonstrate the strength of this source, we measure the two-dimensional spectrum of CdSe quantum dots over a range of population times and find clean signals and clear phonon vibrations. This combination of OPA and hollow-core fiber provides a substantial extension to the capabilities of CMDS.

Antimicrobial Resistance Profile and ExPEC Virulence Potential in Commensal Escherichia coli of Multiple Sources.

We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants.

Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy.

Animals, humans and food are all interconnected sources of antimicrobial resistance (AMR), allowing extensive and rapid exchange of AMR bacteria and genes. Whole genome sequencing (WGS) was used to characterize 279 Escherichia coli isolates obtained from animals (livestock, companion animals, wildlife), food and humans in Italy. E. coli predominantly belonged to commensal phylogroups B1 (46.6%) and A (29%) using the original Clermont criteria. One hundred and thirty-six sequence types (STs) were observed, including different pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Eight antimicrobial resistance genes (ARGs) and five chromosomal mutations conferring resistance to highest priority critically important antimicrobials (HP-CIAs) were identified (qnrS1, qnrB19, mcr-1, blaCTX-M1,15,55, blaCMY-2, gyrA/parC/parE, ampC and pmrB). Twenty-two class 1 integron arrangements in 34 strains were characterized and 11 ARGs were designated as intI1 related gene cassettes (aadA1, aadA2, aadA5, aad23, ant2_Ia, dfrA1, dfrA7, dfrA14, dfrA12, dfrA17, cmlA1). Notably, most intI1 positive strains belonged to rabbit (38%) and poultry (24%) sources. Three rabbit samples carried the mcr-1 colistin resistance gene in association with IS6 family insertion elements. Poultry meat harbored some of the most prominent ExPEC STs, including ST131, ST69, ST10, ST23, and ST117. Wildlife showed a high average number of virulence-associated genes (VAGs) (mean = 10), mostly associated with an ExPEC pathotype and some predominant ExPEC lineages (ST23, ST117, ST648) were identified.

Genomic analysis of trimethoprim-resistant extraintestinal pathogenic Escherichia coli and recurrent urinary tract infections.

Urinary tract infections (UTIs) are the most common bacterial infections requiring medical attention and a leading justification for antibiotic prescription. Trimethoprim is prescribed empirically for uncomplicated cases. UTIs are primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) and ExPEC strains play a central role in disseminating antimicrobial-resistance genes worldwide. Here, we describe the whole-genome sequences of trimethoprim-resistant ExPEC and/or ExPEC from recurrent UTIs (67 in total) from patients attending a regional Australian hospital from 2006 to 2008. Twenty-three sequence types (STs) were observed, with ST131 predominating (28 %), then ST69 and ST73 (both 7 %). Co-occurrence of trimethoprim-resistance genes with genes conferring resistance to extended-spectrum ß-lactams, heavy metals and quaternary ammonium ions was a feature of the ExPEC described here. Seven trimethoprim-resistance genes were identified, most commonly dfrA17 (38 %) and dfrA12 (18 %). An uncommon dfrB4 variant was also observed. Two blaCTX-M variants were identified - blaCTX-M-15 (16 %) and blaCTX-M-14 (10 %). The former was always associated with dfrA12, the latter with dfrA17, and all blaCTX-M genes co-occurred with chromate-resistance gene chrA. Eighteen class 1 integron structures were characterized, and chrA featured in eight structures; dfrA genes featured in seventeen. ST131 H30Rx isolates possessed distinct antimicrobial gene profiles comprising aac(3)-IIa, aac(6)-Ib-cr, aph(3')-Ia, aadA2, blaCTX-M-15, blaOXA-1 and dfrA12. The most common virulence-associated genes (VAGs) were fimH, fyuA, irp2 and sitA (all 91 %). Virulence profile clustering showed ST131 H30 isolates carried similar VAGs to ST73, ST405, ST550 and ST1193 isolates. The sole ST131 H27 isolate carried molecular predictors of enteroaggregative E. coli/ExPEC hybrid strains (aatA, aggR, fyuA). Seven isolates (10 %) carried VAGs suggesting ColV plasmid carriage. Finally, SNP analysis of serial UTI patients experiencing worsening sequelae demonstrated a high proportion of point mutations in virulence factors.

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.

Clonal ST131-H22 Escherichia coli strains from a healthy pig and a human urinary tract infection carry highly similar resistance and virulence plasmids.

The interplay between food production animals, humans and the environment with respect to the transmission of drug-resistant pathogens is widely debated and poorly understood. Pandemic uropathogenic Escherichia coli ST131-H30Rx, with conserved fluoroquinolone and cephalosporin resistance, are not frequently identified in animals. However, the phylogenetic precursor lineage ST131-H22 in animals and associated meat products is being reported with increasing frequency. Here we characterized two highly related ST131-H22 strains, one from a healthy pig and the other from a human infection (in 2007 and 2009, respectively). We used both long and short genome sequencing and compared them to ST131-H22 genome sequences available in public repositories. Even within the context of H22 strains, the two strains in question were highly related, separated by only 20 core SNPs. Furthermore, they were closely related to a faecal strain isolated in 2010 from a geographically distinct, healthy human in New South Wales, Australia. The porcine and hospital strains carried highly similar HI2-ST3 multidrug resistant plasmids with differences in the hospital strain arising due to IS-mediated insertions and rearrangements. Near identical ColV plasmids were also present in both strains, further supporting their shared evolutionary history. This work highlights the importance of adopting a One Health approach to genomic surveillance to gain insights into pathogen evolution and spread.

Whole genome sequence analysis of Australian avian pathogenic Escherichia coli that carry the class 1 integrase gene.

Avian pathogenic Escherichia coli (APEC) cause widespread economic losses in poultry production and are potential zoonotic pathogens. Genome sequences of 95 APEC from commercial poultry operations in four Australian states that carried the class 1 integrase gene intI1, a proxy for multiple drug resistance (MDR), were characterized. Sequence types ST117 (22/95), ST350 (10/95), ST429 and ST57 (each 9/95), ST95 (8/95) and ST973 (7/95) dominated, while 24 STs were represented by one or two strains. FII and FIB repA genes were the predominant (each 93/95, 98 %) plasmid incompatibility groups identified, but those of B/O/K/Z (25/95, 26 %) and I1 (24/95, 25 %) were also identified frequently. Virulence-associated genes (VAGs) carried by ColV and ColBM virulence plasmids, including those encoding protectins [iss (91/95, 96 %), ompT (91/95, 96 %) and traT (90/95, 95 %)], iron-acquisition systems [sitA (88/95, 93 %), etsA (87/95, 92 %), iroN (84/95, 89 %) and iucD/iutA (84/95, 89 %)] and the putative avian haemolysin hylF (91/95, 96 %), featured prominently. Notably, mobile resistance genes conferring resistance to fluoroquinolones, colistin, extended-spectrum ß-lactams and carbapenems were not detected in the genomes of these 95 APEC but carriage of the sulphonamide resistance gene, sul1 (59/95, 63 %), the trimethoprim resistance gene cassettes dfrA5 (48/95, 50 %) and dfrA1 (25/95, 27 %), the tetracycline resistance determinant tet(A) (51/95, 55 %) and the ampicillin resistance genes blaTEM-1A/B/C (48/95, 52 %) was common. IS26 (77/95, 81 %), an insertion element known to capture and mobilize a wide spectrum of antimicrobial resistance genes, was also frequently identified. These studies provide a baseline snapshot of drug-resistant APEC in Australia and their role in the carriage of ColV-like virulence plasmids.

Australian porcine clonal complex 10 (CC10) Escherichia coli belong to multiple sublineages of a highly diverse global CC10 phylogeny.

We recently identified clonal complex 10 (CC10) Escherichia coli as the predominant clonal group in two populations of healthy Australian food-production pigs. CC10 are highly successful, colonizing humans, food-production animals, fresh produce and environmental niches. Furthermore, E. coli within CC10 are frequently drug resistant and increasingly reported as human and animal extra-intestinal pathogens. In order to develop a high-resolution global phylogeny and determine the repertoire of antimicrobial-resistance genes, virulence-associated genes and plasmid types within this clonal group, we downloaded 228 publicly available CC10 short-read genome sequences for comparison with 20 porcine CC10 we have previously described. Core genome single nucleotide polymorphism phylogeny revealed a highly diverse global phylogeny consisting of multiple lineages that did not cluster by geography or source of the isolates. Australian porcine strains belonged to several of these divergent lineages, indicative that CC10 is present in these animals due to multiple colonization events. Differences in resistance gene and plasmid carriage between porcine strains and the global collection highlighted the role of lateral gene transfer in the evolution of CC10 strains. Virulence profiles typical of extra-intestinal pathogenic E. coli were present in both Australian porcine strains and the broader collection. As both the core phylogeny and accessory gene characteristics appeared unrelated to the geography or source of the isolates, it is likely that the global expansion of CC10 is not a recent event and may be associated with faecal carriage in humans.

Whole Genome Sequencing of Escherichia coli From Store-Bought Produce.

The role of agriculture in the transfer of drug resistant pathogens to humans is widely debated and poorly understood. Escherichia coli is a valuable indicator organism for contamination and carriage of antimicrobial resistance (AMR) in foods. Whilst whole genome sequences for E. coli from animals and associated meats are common, sequences from produce are scarce. Produce may acquire drug resistant E. coli from animal manure fertilizers, contaminated irrigation water and wildlife, particularly birds. Whole genome sequencing was used to characterize 120 tetracycline (TET) resistant E. coli from store-bought, ready-to-eat cilantro, arugula and mixed salad from two German cities. E. coli were recovered on the day of purchase and after 7 days of refrigeration. Cilantro was far more frequently contaminated with TET-resistant E. coli providing 102 (85%) sequenced strains. Phylogroup B1 dominated the collection (n = 84, 70%) with multi-locus sequence types B1-ST6186 (n = 37, 31%), C-ST165 (n = 17, 14%), B1-ST58 (n = 14, 12%), B1-ST641 (n = 8, 7%), and C-ST88 (n = 5, 4%) frequently identified. Notably, seven strains of diverse sequence type (ST) carried genetic indicators of ColV virulence plasmid carriage. A number of previously identified and novel integrons associated with insertion elements including IS26 were also identified. Storage may affect the lineages of E. coli isolated, however further studies are needed. Our study indicates produce predominantly carry E. coli with a commensal phylogroup and a variety of AMR and virulence-associated traits. Genomic surveillance of bacteria that contaminate produce should be a matter of public health importance in order to develop a holistic understanding of the environmental dimensions of AMR.

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.

Porcine commensal Escherichia coli: a reservoir for class 1 integrons associated with IS26.

Porcine faecal waste is a serious environmental pollutant. Carriage of antimicrobial-resistance genes (ARGs) and virulence-associated 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.

Tn6026 and Tn6029 are found in complex resistance regions mobilised by diverse plasmids and chromosomal islands in multiple antibiotic resistant Enterobacteriaceae.

Transposons flanked by direct copies of IS26 are important contributors to the evolution of multiple antibiotic resistance. Tn6029 and Tn6026 are examples of composite transposons that have become widely disseminated on small and large plasmids with different incompatibility markers in pathogenic and commensal Escherichia coli and various serovars of Salmonella enterica. Some of the plasmids that harbour these transposons also carry combinations of virulence genes. Recently, Tn6029 and Tn6026 and derivatives thereof have been found on chromosomal islands in both established and recently emerged pathogens. While Tn6029 and Tn6026 carry genes encoding resistance to older generation antibiotics, they also provide a scaffold for the introduction of genes encoding resistance to a wide variety of clinically relevant antibiotics that are mobilised by IS26. As a consequence, Tn6029 and Tn6026 or variants are likely to increasingly feature in complex resistance regions in multiple antibiotic resistant Enterobacteriaceae that threaten the health of humans and food production animals.