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Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD+ hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2’cADPR) and v2-cADPR (3’cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2’cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3’cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3’cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression.One-Sentence SummaryThe chemical structures of two O-glycosidic bond-containing cyclic ADP ribose isomers, the molecular basis of their production, and their function in antiviral and plant immunity suppression by bacteria are reported.

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two β-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogues [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

Bacterial aggregates and biofilms allow bacteria to colonise a diverse array of surfaces that can ultimately lead to infections, where the protection they afford permits bacteria to resist anti-microbials and host immune factors. Despite these advantages there is a trade-off, whereby bacterial spread is reduced. As such, biofilm development needs to be regulated appropriately to suit the required niche. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the formation of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 homologues from diverse pathogenic Escherichia strains. We reveal a common mode of trans-association that leads to cell clumping and show that subtle variations in these interactions governs their aggregation kinetics. Our in depth investigation reveals an underlying molecular basis for the ‘tuning’ of bacterial aggregation.

Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers; however, easy and automated construction of high-quality bacterial genomes remains challenging. Here we present MicroPIPE: a reproducible end-to-end bacterial genome assembly pipeline for ONT and Illumina sequencing. To construct MicroPIPE, we evaluated the performance of several tools for genome reconstruction and assessed overall genome accuracy using ONT both natively and with Illumina. Further validation of MicroPIPE was carried out using 11 sequence type (ST)131 Escherichia coli and eight publicly available Gram-negative and Gram-positive bacterial isolates. MicroPIPE uses Singularity containers and the workflow manager Nextflow and is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe.

Infections caused by carbapenem-resistant Acinetobacter baumannii (CR-Ab) have become increasingly prevalent in clinical settings and often result in significant morbidity and mortality due to their multidrug resistance (MDR). Here we present an integrated whole genome sequencing (WGS) response to a polymicrobial outbreak in a Brisbane hospital between 2016-2018. 28 CR-Ab (and 21 other MDR Gram negative bacilli) were collected from Intensive Care Unit and Burns Unit patients and sent for WGS with a 7-day turn-around-time in clinical reporting. All CR-Ab were sequence type (ST)1050 and within 10 single nucleotide polymorphisms (SNPs) apart, indicative of an ongoing outbreak, and distinct from historical CR-Ab isolates from the same hospital. Possible transmission routes between patients were identified on the basis of CR-Ab and K. pneumoniae SNP profiles. Continued WGS surveillance between 2016 to 2018 enabled suspected outbreak cases to be refuted, but a resurgence of the outbreak CR-Ab mid-2018 in the Burns Unit prompted additional screening. Environmental metagenomic sequencing identified the hospital plumbing as a potential source. Replacement of the plumbing and routine drain maintenance resulted in rapid resolution of the secondary outbreak and significant risk reduction with no discernable transmission in the Burns Unit since. Here we demonstrate implementation of a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.

Escherichia coli Sequence Type (ST)101 is an emerging, multi-drug resistant lineage associated with carbapenem resistance. We recently completed a comprehensive genomics study on mobile genetic elements (MGEs) and their role in blaNDM-1 dissemination within the ST101 lineage. DNA methyltransferases (MTases) are also frequently associated with MGEs, with DNA methylation guiding numerous biological processes including genomic defence against foreign DNA and regulation of gene expression. The availability of Pacific Biosciences Single Molecule Real Time Sequencing data for seven ST101 strains enabled us to investigate the role of DNA methylation on a genome-wide scale (methylome). We defined the methylome of two complete (MS6192 and MS6193) and five draft (MS6194, MS6201, MS6203, MS6204, MS6207) ST101 genomes. Our analysis identified 14 putative MTases and eight N6-methyladenine DNA recognition sites, with one site that has not been described previously. Furthermore, we identified a Type I MTase encoded within a Transposon 7-like Transposon and show its acquisition leads to differences in the methylome between two almost identical isolates. Genomic comparisons with 13 previously published ST101 draft genomes identified variations in MTase distribution, consistent with MGE differences between genomes, highlighting the diversity of active MTases within strains of a single E. coli lineage. It is well established that MGEs can contribute to the evolution of E. coli due to their virulence and resistance gene repertoires. This study emphasises the potential for mobile genetic elements to also enable highly similar bacterial strains to rapidly acquire genome-wide functional differences via changes to the methylome.Impact StatementEscherichia coli ST101 is an emerging human pathogen frequently associated with carbapenem resistance. E. coli ST101 strains carry numerous mobile genetic elements that encode virulence determinants, antimicrobial resistance, and DNA methyltransferases (MTases). In this study we provide the first comprehensive analysis of the genome-wide complement of DNA methylation (methylome) in seven E. coli ST101 genomes. We identified a Transposon carrying a Type I restriction modification system that may lead to functional differences between two almost identical genomes and showed how small recombination events at a single genomic region can lead to global methylome changes across the lineage. We also showed that the distribution of MTases throughout the ST101 lineage was consistent with the presence or absence of mobile genetic elements on which they are encoded. This study shows the diversity of MTases within a single bacterial lineage and shows how strain and lineage-specific methylomes may drive host adaptation.Data SummarySequence data including reads, assemblies and motif summaries have previously been submitted to the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) under the BioProject Accessions: PRJNA580334, PRJNA580336, PRJNA580337, PRJNA580338, PRJNA580339, PRJNA580341 and PRJNA580340 for MS6192, MS6193, MS6194, MS6201, MS6203, MS6204 and MS6207 respectively. All supporting data, code, accessions, and protocols have been provided within the article or through supplementary data files.

BackgroundOXA-48-like carbapenemases have become increasingly prevalent in healthcare settings worldwide. Their low-level activity against carbapenems makes them difficult to identify, causing problems for infection control. Here we present an outbreak of Escherichia coli producing OXA-181 (part of the OXA-48 family of carbapenemases) in a Queensland Hospital, and describe how we used whole genome sequencing (WGS) to identify the outbreak strain, determine the extent of transmission within the hospital and support infection control responses.Methods116 isolates were collected and sequenced on an Illumina NextSeq to determine species, sequence type (ST) and presence of resistance genes. Core single nucleotide polymorphisms were used to determine strain relatedness. Three isolates were also sequenced on an Oxford Nanopore MinION to determine the context of the resistance genes.ResultsOf 116 isolates, 85 (84 E. coli and one K. pneumoniae) from 78 patients (and two environmental sources) were related to the ongoing outbreak. The outbreak E. coli strain was found to be ST38 and carried blaOXA-181, blaCTX-M-15 and qnrS1 genes. Long read sequencing revealed blaOXA-181 to be carried on an IncX3 plasmid with qnrS1. blaCTX-M-15 was chromosomally integrated (via ISEcp1 insertion) in close proximity to a second qnrS1 gene. A search of the laboratory database identified an isolate with an identical unusual antibiogram from a patient recently admitted to a hospital in Vietnam, suggesting that the strain was introduced to the hospital. This conclusion was supported by WGS, as comparison of the strain to public data identified a close match to an E. coli recovered from Vietnam in 2011.ConclusionA blaOXA-181-carrying E. coli ST38 strain was introduced to a Brisbane hospital and spread undetected throughout multiple wards over several months. Using WGS, we characterized the outbreak strain and unambiguously detected its presence throughout the hospital. We show how both WGS and infection control measures can be utilized to effectively terminate widespread transmission of an elusory pathogen.

Carbapenems are last-resort antibiotics; however, the spread of plasmid-encoded carbapenemases such as the New Delhi metallo-β-lactamase 1 (NDM-1) challenges their effectiveness. The rise of NDM-1 has coincided with the emergence of extensively multidrug resistant (MDR) lineages such as Escherichia coli ST101. Here we present a comprehensive genomic analysis of seven E. coli ST101 isolates that carry the blaNDM-1 gene. We determined the complete genomes of two isolates and the draft genomes of five isolates, enabling complete resolution of the plasmid context of blaNDM-1. Comparisons with thirteen previously published ST101 genomes revealed a monophyletic lineage within the B1 phylogroup forming two clades (designated Clade 1 and Clade 2). Most Clade 1 strains are MDR, encoding resistance to at least 9 different antimicrobial classes, including extended spectrum cephalosporins. Additionally, we characterised different pathways for blaNDM-1 carriage and persistence in the ST101 lineage. For IncC plasmids, carriage was associated with recombination and local transposition events within the antibiotic resistance island. In contrast, we revealed recent transfer of a large blaNDM-1 resistance island between F-type plasmids. The complex acquisition pathways characterised here highlight the benefits of long-read Single Molecule Real Time sequencing in revealing evolutionary events that would not be apparent by short-read sequencing alone. These high-quality E. coli ST101 genomes will provide an important reference for further analysis of the role of mobile genetic elements in this emerging multidrug resistant lineage.ImportanceCarbapenem resistant Escherichia coli are urgent priority organisms as they are resistant to our drugs of last resort. E. coli ST101 have been reported as carriers of the New Delhi Metallo-beta-Lactamase 1 gene (blaNDM-1), conferring resistance to carbapenems, however there is limited genomic information available for this lineage. In this study we used long-read genome sequencing to characterise the complete genomes of two E. coli ST101 strains and determine the carriage of blaNDM-1 in a collection of E. coli ST101 strains. We showed that carriage of blaNDM-1 and resistance determinants to eight other antimicrobial classes was confined to a single clade. We also showed two different pathways for the carriage of blaNDM-1, which was dependent on the type of plasmid. Long-read sequencing allowed us to show the full complexities of these resistance regions and highlighted how strains from an emerging E. coli lineage have become resistant to nearly all available antimicrobials.

BackgroundShiga toxigenic Escherichia coli (STEC) is an emerging global pathogen and remains a major cause of food-borne illness with more severe symptoms including hemorrhagic colitis and hemolytic-uremic syndrome. Since the characterization of the archetypal STEC serotype, E. coli O157:H7, more than 250 STEC serotypes have been defined. Many of these non-O157 STEC are associated with clinical cases of equal severity as O157. In this study, we utilize whole genome sequencing of 44 STEC strains from eight serogroups associated with human infection to establish their evolutionary relationships and contrast this with their virulence gene profiles and established typing methods.ResultsOur phylogenomic analysis delineated these STEC strains into seven distinct lineages, each with a characteristic repertoire of virulence factors. Some lineages included commensal or other E. coli pathotypes. Multiple independent acquisitions of the Locus for Enterocyte Effacement were identified, each associated with a distinct repertoire of effector genes. Lineages were inconsistent with O-antigen typing in several instances, consistent with lateral gene transfer within the O-antigen locus. STEC lineages could be defined by the conservation of clustered regularly interspaced short palindromic repeats (CRISPRs), however, no CRISPR profile could differentiate STEC from other E. coli strains. Six genomic regions (ranging from 500 bp - 10 kbp) were found to be conserved across all STEC in this dataset and may dictate interactions with Stx phage lysogeny.ConclusionsThe genomic analyses reported here present non-O157 STEC as a diverse group of pathogenic E. coli emerging from multiple lineages that independently acquired mobile genetic elements that promote pathogenesis.