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Enterotoxigenic Escherichia coli (ETEC) is the primary aetiologic agent of traveller’s diarrhoea and a significant cause of diarrhoeal disease and death in developing countries. ETEC O169:H41 strains are known to cause both traveller’s diarrhoea and foodborne outbreaks in developed countries and are cause for concern. Here, whole-genome sequencing (WGS) was used to assemble 46 O169:H41 (ST182) E. coli draft genomes derived from two airplane waste samples sourced from a German international airport. The ST182 genomes were compared with all 84 publicly available, geographically diverse ST182 genomes to construct a core genome-based phylogenetic tree. ST182 isolates were all phylogroup E, the majority serotype O169:H41 (n = 121, 93%) and formed five major clades. The airplane waste isolates differed by an average of 15 core SNPs (range 0–45) but their accessory genome content was diverse. While uncommon in other ST182 genomes, all airplane-derived ST182 isolates carried: (i) extended-spectrum β-lactamase gene blaCTX–M–15 notably lacking the typical adjacent ISEcp1; (ii) qnrS1 and the S83L mutation in gyrA, both conferring resistance to fluoroquinolones; and (iii) a class 1 integron structure (IS26-intI1Δ648-dfrA17-aadA5-qacEΔ1-sul1-ORF-srpC-padR-IS6100-mphR-mrx-mphA-IS26) identified previously in major extraintestinal pathogenic E. coli STs but not in ETEC. ST182 isolates carried ETEC-specific virulence factors STp + CS6. Adhesin/invasin tia was identified in 89% of aircraft ST182 isolates (vs 23%) and was located on a putative genomic island within a hotspot region for various insertions including PAI I536 and plasmid-associated transposons. The most common plasmid replicons in this collection were IncFII (100%; F2:A-:B-) and IncB/O/K/Z (89%). Our data suggest that potentially through travel, E. coli ST182 are evolving a multidrug-resistant profile through the acquisition of class 1 integrons and different plasmids.

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 PAICL3, 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.

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.

Colistin is a last resort antibiotic used for the treatment of human infections associated with carbapenemase-producing Enterobacteriales. Here, we evaluated the occurrence of mcr-1 and -2 plasmid-mediated colistin resistance in colistin and/or carbapenem resistant human clinical Enterobacteriales and other gram-negative bacteria (n = 543) as well as third generation cephalosporin-resistant (3GCR) Escherichia coli isolates from poultry abattoir workers (n = 15) and poultry fecal samples (n = 92) collected from two geographically separate abattoirs in Romania. which revealed that mcr-1 was present within four sequence types (STs): ST744 (n = 7), ST57 (n = 7), ST156 (n = 2), and ST10 (n = 1). Within STs, serotypes were conserved and, notably, all except one of the mcr-1-positive isolates were found to exhibit fluoroquinolone-resistance (FQR) associated SNPs in both gyrA and parC. While there were variations in genotypes, all isolates belonging to ST744, ST57, and ST156 were rich in resistance determinants, carrying aminoglycoside-modifying enzymes genes, sulfonamide resistance gene blaTEM–1 as well as blaCMY–2 AmpC β-lactamase resistance genes. They also exhibited high similarity in carriage of virulence genes; ST10, however, only carried the mcr-1 gene. Whole genome sequencing (WGS) analysis also revealed that although the mcr-1 gene was identified in a diverse population of E. coli, two STs (ST57 and ST744) predominated and interestingly, were found in isolates across both abattoirs providing evidence for clonal transmission. Also, two main genomic contexts of mcr-1 isolates were revealed with all ST57 isolates harboring the mcr-1 gene between two copies of ISApl1 (or the Tn6330 transposon) whilst a common mcr-1 containing scaffold, highly similar to IncX type mcr-1-bearing plasmids (pWI2-mcr, Accession number: LT838201), was present among mcr-1 isolates of varying phylogenetic backgrounds (ST10, ST744 and ST156). The high prevalence of the mcr-1 gene in poultry E. coli isolates with co-resistance to cephalosporins and quinolones, in a country where antimicrobial use in food production species is poorly regulated, is concerning and the findings from this study should lead to better surveillance of antimicrobial resistance (AMR) in food-production animals in Romania.

Elongation factor thermal unstable Tu (EF-Tu) is a G protein that catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome inside living cells. Structural and biochemical studies have described the complex interactions needed to effect canonical function. However, EF-Tu has evolved the capacity to execute diverse functions on the extracellular surface of both eukaryote and prokaryote cells. EF-Tu can traffic to, and is retained on, cell surfaces where can interact with membrane receptors and with extracellular matrix on the surface of plant and animal cells. Our structural studies indicate that short linear motifs (SLiMs) in surface exposed, non-conserved regions of the molecule may play a key role in the moonlighting functions ascribed to this ancient, highly abundant protein. Here we explore the diverse moonlighting functions relating to pathogenesis of EF-Tu in bacteria and examine putative SLiMs on surface-exposed regions of the molecule.

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.

Escherichia coli ST405 is an emerging urosepsis pathogen, noted for carriage of blaCTX-M, blaNDM, and a repertoire of virulence genes comparable with O25b:H4-ST131. Extraintestinal and multidrug resistant E. coli ST405 are poorly studied in Australia. Here we determined the genome sequence of a uropathogenic, multiple drug resistant E. coli ST405 (strain 2009-27) from the mid-stream urine of a hospital patient in Sydney, Australia, using a combination of Illumina and SMRT sequencing. The genome of strain 2009-27 assembled into two unitigs; a chromosome comprising 5,287,472 bp and an IncB/O plasmid, pSDJ2009-27, of 89,176 bp. In silico and phenotypic analyses showed that strain 2009-27 is a serotype O102:H6, phylogroup D ST405 resistant to ampicillin, azithromycin, kanamycin, streptomycin, trimethoprim, and sulphafurazole. The genes encoding resistance to these antibiotics reside within a novel, mobile IS26-flanked transposon, identified here as Tn6242, in the chromosomal gene yjdA. Tn6242 comprises four modules that each carries resistance genes flanked by IS26, including a class 1 integron with dfrA17 and aadA5 gene cassettes, a variant of Tn6029, and mphA. We exploited unique genetic signatures located within Tn6242 to identify strains of ST405 from Danish patients that also carry the transposon in the same chromosomal location. The acquisition of Tn6242 into yjdA in ST405 is significant because it (i) is vertically inheritable; (ii) represents a reservoir of resistance genes that can transpose onto resident/circulating plasmids; and (iii) is a site for the capture of further IS26-associated resistance gene cargo.

Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

Full genome sequences of 20 strains of Clostridium chauvoei, the etiological agent of blackleg of cattle and sheep, isolated from four different continents over a period of 64 years (1951–2015) were determined and analyzed. The study reveals that the genome of the species C. chauvoei is highly homogeneous compared to the closely related species C. perfringens, a widespread pathogen that affects human and many animal species. Analysis of the CRISPR locus is sufficient to differentiate most C. chauvoei strains and is the most heterogenous region in the genome, containing in total 187 different spacer elements that are distributed as 30 – 77 copies in the various strains. Some genetic differences are found in the 3 allelic variants of fliC1, fliC2 and fliC3 genes that encode structural flagellin proteins, and certain strains do only contain one or two alleles. However, the major virulence genes including the highly toxic C.chauvoei toxin A, the sialidase and the two hyaluronidases are fully conserved as are the metabolic and structural genes of C. chauvoei. These data indicate that C. chauvoei is a strict ruminant-associated pathogen that has reached a dead end in its evolution.

The discovery of antibiotics heralded the start of a ‘Golden Age’ in the history of medicine. Over the years, the use of antibiotics extended beyond medical practice into animal husbandry, aquaculture and agriculture. While the ‘Golden Age’ featured the rapid discovery of a series of new classes of antibiotics, the current discovery/development pipeline has been less productive. We are now faced with the threat of diseases mediated by pathogenic bacteria that are resistant to all major classes of antibiotic. Evolution of bacteria resistant to multiple antibiotics results from the inherent genetic capability that bacteria have to adapt rapidly to changing environmental conditions. Consequently, under antibiotic selection pressures, bacteria have acquired resistance to all classes of antibiotics, sometimes very shortly after their introduction. Multiple drug resistant (MDR) bacteria are found worldwide and are possibly signalling the end of the antibiotic era. Arguably, the evolution and rapid dissemination of MDR genes en-masse across microbial pathogens is one of the most serious threats to human health. In this context, effective surveillance strategies to track the development of resistance to multiple antibiotics are vital to managing global infection control. These surveillance strategies are necessary for not only human health but also for animal health and plant production. Shortfalls in the present surveillance strategies need to be identified. Raising awareness of the genetic events that promote co-selection of resistance to multiple antimicrobials is an important prerequisite to the design and implementation of molecular surveillance strategies. In this review we will discuss how surveillance strategies are important for our understanding of the possible relationship between the use of low-dose antibiotics in animal husbandry and evolution of MDR in Gram-negative bacteria.

Multiple antibiotic resistant pathogens represent a major clinical challenge in both the human and veterinary context. It is now well understood that the genes that encode resistance are context independent. That is, the same gene is commonly present in otherwise very disparate pathogens in both humans and production and companion animals, and among bacteria that proliferate in an agricultural context. This can be true even for pathogenic species or clonal types that are otherwise confined to a single host or ecological niche. It therefore follows that mechanisms of gene flow must exist to move genes from one part of the microbial biosphere to another. It is widely accepted that lateral (or horizontal) gene transfer (L(H)GT) drives this gene flow. LGT is relatively well understood mechanistically but much of this knowledge is derived from a reductionist perspective. We believe that this is impeding our ability to deal with the medical ramifications of LGT. Resistance genes and the genetic scaffolds that mobilize them in MDR bacteria of clinical significance are likely to have their origins in completely unrelated parts of the microbial biosphere. Resistance genes are increasingly polluting the microbial biosphere by contaminating environmental niche where previously they were not detected. More attention needs to be paid to the way that humans have, through the widespread application of antibiotics, selected for combinations of mobile elements that enhance the flow of resistance genes between remotely linked parts of the microbial biosphere. Attention also needs to be paid to those bacteria that link human and animal ecosystems. We argue that multiply antibiotic resistant commensal bacteria are especially important in this regard. More generally, the post genomics era offers the opportunity for understanding how resistance genes are mobilized from a one health perspective. In the long term, this holistic approach offers the best opportunity to better manage wh