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Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs.

In virtually all eukaryotes, the mitochondrial genome (mitochondrial DNA, mtDNA) encodes proteins necessary for oxidative phosphorylation (OXPHOS) and the RNA machinery required for their synthesis inside the mitochondria. Appropriate regulation of mtDNA copy number and expression is essential for ensuring the correct stoichiometric formation of OXPHOS complexes assembled from both nuclear- and mtDNA-encoded subunits. The mechanisms of mtDNA regulation are not completely understood but are essential to organismal viability and lifespan. Here, using multiple approaches, we identify the presence of N6-methylation (6mA) on the mtDNA of diverse animal and plant species. Importantly, we also demonstrate that this modification is regulated inC. elegansby the DNA methyltransferase DAMT-1, and DNA demethylase ALKB-1, which localize to mitochondria. Misregulation of mtDNA 6mA through targeted overexpression of these enzymatic activities inappropriately alters mtDNA copy number and expression, impairing OXPHOS function and producing increased oxidative stress, as well as a shortened lifespan. Compounding defects in mtDNA regulation, reductions in mtDNA 6mA methylation promote the propagation of a deleterious mitochondrial genome across generations. Together, these results reveal that mtDNA 6mA is highly conserved among eukaryotes and regulates lifespan by influencing mtDNA copy number, expression, and heritable mutation levelsin vivo.

We report a fatal case of Aspergillus felis invasive rhinosinusitis with secondary cerebral abscesses in an immunocompetent host despite aggressive surgical debridement and combination antifungals. Whilst this organism is known to cause fatalities in cats, only a few cases in humans have been documented, all of which had significant immunosuppression. This is the first human death due to A. felis described in an immunocompetent host.

The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing togt;95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g.,iElizabethkingia/i) and some β-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.

BackgroundA prerequisite to rapid molecular detection of pathogens causing bloodstream infections is an efficient, cost effective and robust DNA extraction solution. We describe methods for microbial DNA extraction direct from positive blood culture broths, suitable for metagenomic sequencing and the application of machine-learning based tools to predict antimicrobial susceptibility.MethodsProspectively collected culture-positive blood culture broths with Gram-negative bacteria, were directly extracted using various commercially available kits. We compared methods for efficient inhibitor removal, avoidance of DNA shearing or degradation, to achieve DNA of high quality and purity. Bacterial species identified via whole-genome metagenomic sequencing (Illumina, MiniSeq) from blood culture extracts were compared to conventional methods from cultured isolates (Vitek MS). A machine-learning algorithm (AREScloud) was used to predict susceptibility against commercially available antibiotics, compared to susceptibility testing (Vitek 2) and other commercially available rapid diagnostic instruments (Accelerate Pheno and BCID).ResultsA two-kit method using a modified MolYsis Basic kit (for host DNA depletion) and extraction using Qiagen DNeasy UltraClean microbial kits resulted in optimal extractions appropriate for multiple molecular applications, including PCR, short-read and long-read sequencing. DNA extracts from 40 blood culture broths were included. Taxonomic profiling by direct metagenomic sequencing matched species identification by conventional methods in 38/40 (95%) of samples, with two showing agreement to genus level. In two polymicrobial samples, a second organism was missed by sequencing. Whole genome sequencing antimicrobial susceptibility testing (WGS-AST) models were able to accurately infer profiles for 6 common pathogens against 17 antibiotics. Overall categorical agreement (CA) was 95%, with 11% very major errors (VME) and 3.9% major errors (ME). CA for WGS-AST was >95% for 5/6 of the most common pathogens (E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and C. jejuni) while it was lower for K. oxytoca (66.7%), likely due to the presence of inducible cephalosporinases. Performance of WGS-AST was sub-optimal for uncommon pathogens (e.g. Elizabethkingia) and some combination antibiotic compounds (e.g. ticarcillin-clavulanate). Time to pathogen identification and resistance gene detection was fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a rapid MIC result in approximately 8 h. While Illumina based direct metagenomic sequencing did not result in faster turn-around times compared conventional methods, use of real-time nanopore sequencing may allow faster data acquisition.ConclusionsThe application of direct metagenomic sequencing from positive blood culture broths is a feasible approach and solves some of the challenges of sequencing from low-bacterial load samples. Machine-learning based algorithms are also accurate for common pathogen / drug combinations, although additional work is required to optimise algorithms for uncommon species and more complex resistance genotypes, as well as streamlining methods to provide more rapid sequencing results.

The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens1-3. Although aspects of IncC plasmid conjugation have been well studied4-9, many roles of conjugation genes have been assigned based solely on sequence similarity. We applied hypersaturated transposon mutagenesis and transposon-directed insertion-site sequencing to determine the set of genes required for IncC conjugation. We identified 27 conjugation genes, comprising 19 that were previously identified (including two regulatory genes, acaDC) and eight not previously associated with conjugation. We show that one previously unknown gene, acaB, encodes a transcriptional regulator that has a crucial role in the regulation of IncC conjugation. AcaB binds upstream of the acaDC promoter to increase acaDC transcription; in turn, AcaDC activates the transcription of IncC conjugation genes. We solved the crystal structure of AcaB at 2.9-A resolution and used this to guide functional analyses that reveal how AcaB binds to DNA. This improved understanding of IncC conjugation provides a basis for the development of new approaches to reduce the spread of these multi-drug-resistance plasmids.

BackgroundProspective whole-genome sequencing (WGS)-based surveillance may be the optimal approach to rapidly identify transmission of multi-drug resistant (MDR) bacteria in the healthcare setting.Materials/methodsWe prospectively collected methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenem-resistant Acinetobacter baumannii (CRAB), extended-spectrum beta-lactamase (ESBL-E) and carbapenemase-producing Enterobacterales (CPE) isolated from blood cultures, sterile sites or screening specimens across three large tertiary referral hospitals (2 adult, 1 paediatric) in Brisbane, Australia. WGS was used to determine in silico multi-locus sequence typing (MSLT) and resistance gene profiling via a bespoke genomic analysis pipeline. Putative transmission events were identified by comparison of core genome single nucleotide polymorphisms (SNPs). Relevant clinical meta-data were combined with genomic analyses via customised automation, collated into hospital-specific reports regularly distributed to infection control teams.ResultsOver four years (April 2017 to July 2021) 2,660 isolates were sequenced. This included MDR gram-negative bacilli (n=293 CPE, n=1309 ESBL), MRSA (n=620) and VRE (n=433). A total of 379 clinical reports were issued. Core genome SNP data identified that 33% of isolates formed 76 distinct clusters. Of the 76 clusters, 43 were contained to the three target hospitals, suggesting ongoing transmission within the clinical environment. The remaining 33 clusters represented possible inter-hospital transmission events or strains circulating in the community. In one hospital, proven negligible transmission of non-multi-resistant MRSA enabled changes to infection control policy.ConclusionsImplementation of routine WGS for MDR pathogens in clinical laboratories is feasible and can enable targeted infection prevention and control interventions.SummaryWe initiated a program of routine sequencing of multi-drug resistant organisms. A custom analysis pipeline was used to automate reporting by incorporating clinical meta-data with genomics to define clusters and support infection control interventions.

Clinical and public health microbiology is increasingly utilising whole genome sequencing (WGS) technology and this has lead to the development of a myriad of analysis tools and bioinformatics pipelines. Single nucleotide polymorphism (SNP) analysis is an approach used for strain characterisation and determining isolate relatedness. However, in order to ensure the development of robust methodologies suitable for clinical application of this technology, accurate, reproducible, traceable and benchmarked analysis pipelines are necessary. To date, the approach to benchmarking of these has been largely ad-hoc with new pipelines benchmarked on their own datasets with limited comparisons to previously published pipelines.In this study, Snpdragon, a fast and accurate SNP calling pipeline is introduced. Written in Nextflow, Snpdragon is capable of handling small to very large and incrementally growing datasets. Snpdragon is benchmarked using previously published datasets against six other all-in-one microbial SNP calling pipelines, Lyveset, Lyveset2, Snippy, SPANDx, BactSNP and Nesoni. The effect of dataset choice on performance measures is demonstrated to highlight some of the issues associated with the current available benchmarking approaches.The establishment of an agreed upon gold-standard benchmarking process for microbial variant analysis is becoming increasingly important to aid in its robust application, improve transparency of pipeline performance under different settings and direct future improvements and development.Snpdragon is available at https://github.com/FordeGenomics/SNPdragon.Impact statementWhole-genome sequencing has become increasingly popular in infectious disease diagnostics and surveillance. The resolution provided by single nucleotide polymorphism (SNP) analyses provides the highest level of insight into strain characteristics and relatedness. Numerous approaches to SNP analysis have been developed but with no established gold-standard benchmarking approach, choice of bioinformatics pipeline tends to come down to laboratory or researcher preference. To support the clinical application of this technology, accurate, transparent, auditable, reproducible and benchmarked pipelines are necessary. Therefore, Snpdragon has been developed in Nextflow to allow transparency, auditability and reproducibility and has been benchmarked against six other all-in-one pipelines using a number of previously published benchmarking datasets. The variability of performance measures across different datasets is shown and illustrates the need for a robust, fair and uniform approach to benchmarking.Data SummaryPreviously sequenced reads for Escherichia coli O25b:H4-ST131 strain EC958 are available in BioProject PRJNA362676. BioSample accession numbers for the three benchmarking isolates are:EC958: SAMN06245884MS6573: SAMN06245879MS6574: SAMN06245880Accession numbers for reference genomes against the E. coli O25b:H4-ST131 strain EC958 benchmark are detailed in table 2.Simulated benchmarking data previously described by Yoshimura et al. is available at http://platanus.bio.titech.ac.jp/bactsnp (1).Simulated datasets previously described by Bush et al. is available at http://dx.doi.org/10.5287/bodleian:AmNXrjYN8 (2).Real sequencing benchmarking datasets previously described by Bush et al. are available at http://dx.doi.org/10.5287/bodleian:nrmv8k5r8 (2).

Fowl cholera caused by

Objectives To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. Methods For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (n = 10) and non-gonococcal (n = 5) isolates and a panel of N. gonorrhoeae nucleic acid amplification test (NAAT)-positive clinical sample extracts (n = 40). Results The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical sample extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical samples confirmed to have WT gyrA sequences based on Sanger sequencing. Conclusions These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO.

Fish mortality caused by

Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa-positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/ Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.

Escherichia coli ST131 is a recently emerged antibiotic resistant clone responsible for high rates of urinary tract and bloodstream infections. Despite its global dominance, the precise mechanisms that have driven the rapid dissemination of ST131 remain unknown. Here, we show that the plasmid-associated resistance gene encoding the AAC(6’)-Ib-cr enzyme that inactivates the fluoroquinolone (FQ) antibiotic ciprofloxacin is present in >70% of strains from the most rapidly expanding subgroup of multidrug resistant ST131. Using a series of genome-edited and plasmid-cured isogenic strains, we demonstrate that the aac(6’)-Ib-cr gene confers a selective advantage on ST131 in the presence of ciprofloxacin, even in strains containing chromosomal GyrA and ParC FQ-resistance mutations. Further, we identify a pattern of emerging carbapenem resistance in other common E. coli clones carrying both aac(6’)-Ib-cr and chromosomal FQ-resistance mutations, suggesting this dual resistance combination may also impart a selective advantage on these non-ST131 antibiotic resistant lineages.

Background Urosepsis caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is increasing worldwide. Carbapenems are commonly recommended for the treatment of ESBL infections; however, to minimize the emergence of carbapenem resistance, interest in alternative treatments has heightened. Objectives This study compared pharmacodynamics of piperacillin/tazobactam versus meropenem against ESBL-producing and non-producing E. coli clinical isolates. Methods E. coli isolates, obtained from national reference laboratory in Bangladesh, were characterized by phenotypic tests, WGS, susceptibility tests and mutant frequency analysis. Three ESBL-producing and two non-producing E. coli were exposed to piperacillin/tazobactam (4.5 g, every 6 h and every 8 h, 30 min infusion) and meropenem (1 g, every 8 h, 30 min infusion) in a hollow-fibre infection model over 7 days. Results Piperacillin/tazobactam regimens attained ∼4–5 log10 cfu/mL bacterial killing within 24 h and prevented resistance emergence over the experiment against ESBL-producing and non-producing E. coli. However, compared with 8 hourly meropenem, the 6 hourly piperacillin/tazobactam attained ∼1 log10 lower bacterial kill against one of three ESBL-producing E. coli (CTAP#173) but comparable killing for the other two ESBL-producing (CTAP#168 and CTAP#169) and two non-producing E. coli (CTAP#179 and CTAP#180). The 6 hourly piperacillin/tazobactam regimen attained ∼1 log10 greater bacterial kill compared with the 8 hourly regimen against CTAP#168 and CTAP#179 at 24 h. Conclusions Our study suggests piperacillin/tazobactam may be a potential alternative to carbapenems to treat urosepsis caused by ESBL-producing E. coli, although clinical trials with robust design are needed to confirm non-inferiority of outcome.

Motivation Whole genome sequencing (WGS) is revolutionizing disease surveillance where it facilitates high-resolution clustering of related organism and outbreak detection. However, visualizing and efficiently communicating genomic data back to clinical staff is crucial for the successful deployment of a targeted infection control response. Results CATHAI (cluster analysis tool for healthcare-associated infections) is an interactive web-based visualization platform that couples WGS informed clustering with associated metadata, thereby converting sequencing data into informative and accessible clinical information for the management of healthcare-associated infections (HAI) and nosocomial outbreaks. Availability and implementation All code associated with this application are free available from https://github.com/FordeGenomics/cathai. A demonstration version of CATHAI is available online at https://cathai.fordelab.com.

INTRODUCTION : Antimicrobial resistance (AMR) continues to present major challenges to modern healthcare. Recent advances in whole genome sequencing (WGS) have made the rapid molecular characterization of AMR a realistic possibility for diagnostic laboratories; yet major barriers to clinical implementation exist. AREAS COVERED : We describe and compare short- and long-read sequencing platforms, typical components of bioinformatics pipelines, tools for AMR gene detection and the relative merits of read- or assembly-based approaches. The challenges of characterizing mobile genetic elements from genomic data are outlined, as well as the complexities inherent to the prediction of phenotypic resistance from WGS. Practical obstacles to implementation in diagnostic laboratories, the critical role of quality control and external quality assurance, as well as standardized reporting standards are also discussed. Future directions, such as the application of machine-learning and artificial intelligence algorithms, linked to clinically meaningful outcomes, may offer a new paradigm for the clinical application of AMR prediction. EXPERT OPINION : AMR prediction from WGS data presents an exciting opportunity to advance our capacity to comprehensively characterize infectious pathogens in a rapid manner, ultimately aiming to improve patient outcomes. Collaborative efforts between clinicians, scientists, regulatory bodies and healthcare administrators will be critical to achieve the full promise of this approach.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are a global public-health concern. We evaluated the pharmacodynamic activity of piperacillin/tazobactam (TZP) dosing regimens against ESBL-producing versus non-ESBL-producing E. coli. Five E. coli clinical isolates were obtained from Bangladesh. Broth microdilution and whole-genome sequencing (WGS) were performed on the five studied isolates. Three TZP-susceptible ESBL-producing and two non-ESBL-producing E. coli were exposed to TZP regimens of 4.5 g every 6 h (q6h) and every 8 h (q8h) as a 30-min infusion in a dynamic hollow-fibre infection model over 7 days. The extent of bacterial killing was ∼4-5 log

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure., Recurrent urinary tract infections occur in ~ 25% of women. Here, Beatson and colleagues use whole genome sequencing to track the dynamics of an E. coli ST131 clone in a single patient over a 5-year period. This study provides unique insights into pathogen evolution during recurrent urinary infection.

Background Carbapenems are recommended treatment for serious infections caused by AmpC-producing gram-negative bacteria but can select for carbapenem resistance. Piperacillin-tazobactam may be a suitable alternative. Methods We enrolled adult patients with bloodstream infection due to chromosomal AmpC producers in a multicenter randomized controlled trial. Patients were assigned 1:1 to receive piperacillin-tazobactam 4.5 g every 6 hours or meropenem 1 g every 8 hours. The primary efficacy outcome was a composite of death, clinical failure, microbiological failure, and microbiological relapse at 30 days. Results Seventy-two patients underwent randomization and were included in the primary analysis population. Eleven of 38 patients (29%) randomized to piperacillin-tazobactam met the primary outcome compared with 7 of 34 patients (21%) in the meropenem group (risk difference, 8% [95% confidence interval {CI}, –12% to 28%]). Effects were consistent in an analysis of the per-protocol population. Within the subcomponents of the primary outcome, 5 of 38 (13%) experienced microbiological failure in the piperacillin-tazobactam group compared to 0 of 34 patients (0%) in the meropenem group (risk difference, 13% [95% CI, 2% to 24%]). In contrast, 0% vs 9% of microbiological relapses were seen in the piperacillin-tazobactam and meropenem arms, respectively. Susceptibility to piperacillin-tazobactam and meropenem using broth microdilution was found in 96.5% and 100% of isolates, respectively. The most common AmpC β-lactamase genes identified were blaCMY-2, blaDHA-17, blaCMH-3, and blaACT-17. No ESBL, OXA, or other carbapenemase genes were identified. Conclusions Among patients with bloodstream infection due to AmpC producers, piperacillin-tazobactam may lead to more microbiological failures, although fewer microbiological relapses were seen. Clinical Trials Registration NCT02437045.

Background Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing. Results We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes. Conclusions MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe.

BackgroundSepsis contributes significantly to morbidity and mortality globally. In Australia, 20,000 develop sepsis every year, resulting in 5,000 deaths, and more than AUD$846 million in expenditure. Prompt, appropriate antibiotic therapy is effective in improving outcomes in sepsis. Conventional culture-based methods to identify appropriate therapy have limited yield and take days to complete. Recently, nanopore technology has enabled rapid sequencing with real-time analysis of pathogen DNA. We set out to demonstrate the feasibility and diagnostic accuracy of pathogen sequencing direct from clinical samples, and estimate the impact of this approach on time to effective therapy when integrated with personalised software-guided antimicrobial dosing in children and adults on ICU with sepsis.MethodsThe DIRECT study is a pilot prospective, non-randomized multicentre trial of an integrated diagnostic and therapeutic algorithm combining rapid direct pathogen sequencing and software-guided, personalised antibiotic dosing in children and adults with sepsis on ICU.Participants and interventionsDIRECT will collect microbiological and pharmacokinetic samples from approximately 200 children and adults with sepsis admitted to one of four ICUs in Brisbane. In Phase 1, we will evaluate Oxford Nanopore Technologies MinION sequencing direct from blood in 50 blood culture-proven sepsis patients recruited from consecutive patients with suspected sepsis. In Phase 2, a further 50 consecutive patients with suspected sepsis will be recruited in whom MinION sequencing will be combined with Bayesian software-guided (ID-ODS) personalised antimicrobial dosing.Outcome measuresThe primary outcome is time to effective antimicrobial therapy, defined as trough drug concentrations above the MIC of the pathogen. Secondary outcomes are diagnostic accuracy of MinION sequencing from whole blood, time to pathogen identification and susceptibility testing using sequencing direct from whole blood and from positive blood culture broth.DiscussionRapid pathogen sequencing coupled with antimicrobial dosing software has great potential to overcome the limitations of conventional diagnostics which often result in prolonged inappropriate antimicrobial therapy. Reduced time to optimal antimicrobial therapy may reduce sepsis mortality and ICU length of stay. This pilot study will yield key feasibility data to inform further, urgently needed sepsis studies. Phase 2 of the trial protocol is registered with the ANZCTR (ACTRN12620001122943).Trial registrationRegistered with the Australia New Zealand Clinical Trials Registry Number ACTRN12620001122943

In this study, we aimed to investigate the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) clinical and colonizing isolates of dogs and cats to profile contributing factors associated with their isolation. Nasal and rectal samples were collected from dogs and cats between 2015 and 2017 to identify colonizing isolates. Clinical isolates collected between 2003 and 2016 were retrieved from a Queensland university veterinary diagnostic laboratory. All isolates were identified using standard microbiological and molecular methods and were characterized by whole genome sequencing. Phylogenetic relationships and differences in epidemiological factors were investigated. Seventy-two MRSP isolates out of 1,460 colonizing samples and nine MRSP clinical isolates were identified. No MRSA was isolated. ST496 and ST749 were the most commonly isolated sequence types with different SCCmec types. ST496 clones spread both along the coast and more inland where ST749 was more centered in Brisbane. The resistance and virulence factors differed significantly between the two sequence types. ST496 colonizing and clinical isolates were similarly multidrug resistant. The virulence genes of ST749 colonizing and clinical isolates were similar as both contained the gene nanB for sialidase. There were no differences in the individual and clinical factors between predominant sequence types. High levels of antimicrobial resistance occurred in the majority of isolates, which is of potential concern to human and veterinary health. The phylogenetic clustering of isolates from this study and others previously identified in countries, particularly New Zealand, with which Australia has high volume of pet movements could suggest the importation of clones, which needs further investigation.

Background. 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 persistent CR-Ab outbreak in a Brisbane hospital between 2016–2018. Methods. A. baumannii, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa isolates were sequenced using the Illumina platform primarily to establish isolate relationships based on core-genome SNPs, MLST and antimicrobial resistance gene profiles. Representative isolates were selected for PacBio sequencing. Environmental metagenomic sequencing with Illumina was used to detect persistence of the outbreak strain in the hospital. Results. In response to a suspected polymicrobial outbreak between May to August of 2016, 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 (Pasteur ST2) and within 10 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. Conclusion. We implemented a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.

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.

ObjectiveTo predict the cost and health effects of routine use of whole-genome sequencing (WGS) of bacterial pathogens compared with those of standard of care.DesignBudget impact analysis was performed over the following 5 years. Data were primarily from sequencing results on clusters of multidrug-resistant organisms across 27 hospitals. Model inputs were derived from hospitalisation and sequencing data, and epidemiological and costing reports, and included multidrug resistance rates and their trends.SettingQueensland, Australia.ParticipantsHospitalised patients.InterventionsWGS surveillance of six common multidrug-resistant organisms (Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter sp and Acinetobacter baumannii) compared with standard of care or routine microbiology testing.Primary and secondary outcomesExpected hospital costs, counts of patient infections and colonisations, and deaths from bloodstream infections.ResultsIn 2021, 97 539 patients in Queensland are expected to be infected or colonised with one of six multidrug-resistant organisms with standard of care testing. WGS surveillance strategy and earlier infection control measures could avoid 36 726 infected or colonised patients and avoid 650 deaths. The total cost under standard of care was $A170.8 million in 2021. WGS surveillance costs an additional $A26.8 million but was offset by fewer costs for cleaning, nursing, personal protective equipment, shorter hospital stays and antimicrobials to produce an overall cost savings of $30.9 million in 2021. Sensitivity analyses showed cost savings remained when input values were varied at 95% confidence limits.ConclusionsCompared with standard of care, WGS surveillance at a state-wide level could prevent a substantial number of hospital patients infected with multidrug-resistant organisms and related deaths and save healthcare costs. Primary prevention through routine use of WGS is an investment priority for the control of serious hospital-associated infections.

In this study, we aimed to investigate the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) clinical and colonizing isolates of dogs and cats to profile contributing factors associated with their isolation. Nasal and rectal samples were collected from dogs and cats between 2015 and 2017 to identify colonizing isolates. Clinical isolates collected between 2003 and 2016 were retrieved from a Queensland university veterinary diagnostic laboratory. All isolates were identified using standard microbiological and molecular methods and were characterized by whole genome sequencing. Phylogenetic relationships and differences in epidemiological factors were investigated. Seventy-two MRSP isolates out of 1,460 colonizing samples and nine MRSP clinical isolates were identified. No MRSA was isolated. ST496 and ST749 were the most commonly isolated sequence types with different SCCmec types. ST496 clones spread both along the coast and more inland where ST749 was more centered in Brisbane. The resistance and virulence factors differed significantly between the two sequence types. ST496 colonizing and clinical isolates were similarly multidrug resistant. The virulence genes of ST749 colonizing and clinical isolates were similar as both contained the gene nanB for sialidase. There were no differences in the individual and clinical factors between predominant sequence types. High levels of antimicrobial resistance occurred in the majority of isolates, which is of potential concern to human and veterinary health. The phylogenetic clustering of isolates from this study and others previously identified in countries, particularly New Zealand, with which Australia has high volume of pet movements could suggest the importation of clones, which needs further investigation.

Despite the accepted dogma that antibiotic use is the largest contributor to antimicrobial resistance (AMR) and human microbiome disruption, our knowledge of specific antibiotic-microbiome effects remains basic. Detection of associations between new or old antimicrobials and specific AMR burden is patchy and heterogeneous. Various microbiome analysis tools are available to determine antibiotic effects on microbial communities in vivo. Microbiome analysis of treatment groups in antibiotic clinical trials, powered to measure clinically meaningful endpoints would greatly assist the antibiotic development pipeline and clinician antibiotic decision making.

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.

Highlights • Two different disease manifestations in 2 fur seals associated with P. multocida. • Whole genome sequencing revealed the presence of within-host diversity of P. multocida, a novel finding for this important pathogen. • The isolates from several internal organs of the two seals belonged to the same genetic lineage. • Results suggest that transiting birds could have been responsible for introducing the outbreak strain., Pasteurella multocida is a heterogeneous bacterium, which has the capacity to cause disease in a wide range of host species and is also recognized as an important zoonotic pathogen. Two sequential deaths in captive fur seals occurred at Sea World, Australia during December 2017. A fibrinosuppurative bronchopneumonia in a Subantarctic fur seal (Arctocephalus tropicalis) resulted in death within 24 h of nonspecific signs of illness, whereas a septic peritonitis in a New Zealand fur seal (Arctocephalus forsteri) resulted in death within 12 h of clinical presentation. The cases happened within three days in two different pool locations, although both had previously been housed in the same area. A total of six Pasteurella multocida isolates were obtained from several internal organs at necropsy in both cases and were subjected to whole genome sequencing and phylogenomic analysis. In-silico typing of the isolates revealed that all belonged to Multi-Locus Sequence Type 7 and carried lipopolysaccharide outer core biosynthesis loci Type 3. Phylogenomic analysis of the isolates confirmed that the isolates were near identical at the core genome level, suggesting acquisition from a common source. The results also revealed the presence of within host and across animal diversity of P. multocida isolates for the first time even in a clearly connected outbreak.

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.

Antimicrobial-resistant ESKAPE ( E nterococcus faecium , S taphylococcus aureus , K lebsiella pneumoniae , A cinetobacter baumannii , P seudomonas aeruginosa , and E nterobacter species) pathogens represent a global threat to human health. The acquisition of antimicrobial resistance genes by ESKAPE pathogens has reduced the treatment options for serious infections, increased the burden of disease, and increased death rates due to treatment failure and requires a coordinated global response for antimicrobial resistance surveillance.

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

Background Diphtheria is a potentially fatal respiratory disease caused by toxigenic Corynebacterium diphtheriae. Although resistance to erythromycin has been recognized, β-lactam resistance in toxigenic diphtheria has not been described. Here, we report a case of fatal respiratory diphtheria caused by toxigenic C. diphtheriae resistant to penicillin and all other β-lactam antibiotics, and describe a novel mechanism of inducible carbapenem resistance associated with the acquisition of a mobile resistance element. Methods Long-read whole-genome sequencing was performed using Pacific Biosciences Single Molecule Real-Time sequencing to determine the genome sequence of C. diphtheriae BQ11 and the mechanism of β-lactam resistance. To investigate the phenotypic inducibility of meropenem resistance, short-read sequencing was performed using an Illumina NextSeq500 sequencer on the strain both with and without exposure to meropenem. Results BQ11 demonstrated high-level resistance to penicillin (benzylpenicillin minimum inhibitory concentration [MIC] ≥ 256 μg/ml), β-lactam/β-lactamase inhibitors and cephalosporins (amoxicillin/clavulanic acid MIC ≥ 256 μg/mL; ceftriaxone MIC ≥ 8 μg/L). Genomic analysis of BQ11 identified acquisition of a novel transposon carrying the penicillin-binding protein (PBP) Pbp2c, responsible for resistance to penicillin and cephalosporins. When strain BQ11 was exposed to meropenem, selective pressure drove amplification of the transposon in a tandem array and led to a corresponding change from a low-level to a high-level meropenem-resistant phenotype. Conclusions We have identified a novel mechanism of inducible antibiotic resistance whereby isolates that appear to be carbapenem susceptible on initial testing can develop in vivo resistance to carbapenems with repeated exposure. This phenomenon could have significant implications for the treatment of C. diphtheriae infection, and may lead to clinical failure.

Polymyxins remain one of the last-resort drugs to treat infections caused by MDR Gram-negative pathogens. Here, we determined the mechanisms by which chromosomally encoded resistance to colistin and polymyxin B can arise in the MDR uropathogenic Escherichia coli ST131 reference strain EC958. Two complementary approaches, saturated transposon mutagenesis and spontaneous mutation induction with high concentrations of colistin and polymyxin B, were employed to select for mutations associated with resistance to polymyxins. Mutants were identified using transposon-directed insertion-site sequencing or Illumina WGS. A resistance phenotype was confirmed by MIC and further investigated using RT-PCR. Competitive growth assays were used to measure fitness cost. A transposon insertion at nucleotide 41 of the pmrB gene (EC958pmrB41-Tn5) enhanced its transcript level, resulting in a 64- and 32-fold increased MIC of colistin and polymyxin B, respectively. Three spontaneous mutations, also located within the pmrB gene, conferred resistance to both colistin and polymyxin B with a corresponding increase in transcription of the pmrCAB genes. All three mutations incurred a fitness cost in the absence of colistin and polymyxin B. This study identified the pmrB gene as the main chromosomal target for induction of colistin and polymyxin B resistance in E. coli.

Fowl cholera, caused by Pasteurella multocida, continues to be a challenge in meat-chicken-breeder operations and has emerged as a problem for free-range meat chickens. Here, using whole-genome sequencing (WGS) and phylogenomic analysis, we investigate isolate relatedness during outbreaks of fowl cholera on a free-range meat chicken farm over a 5-year period. Our genomic analysis revealed that while all outbreak isolates were sequence type (ST) 20, they could be separated into two distinct clades (clade 1 and clade 2) consistent with difference in their lipopolysaccharide (LPS) type. The isolates from the earlier outbreaks (clade 1) were carrying LPS type L3 while those from the more recent outbreaks (clade 2) were LPS type L1. Additionally, WGS data indicated high inter- and intra-chicken genetic diversity during a single outbreak. Furthermore, we demonstrate that while a killed autogenous vaccine carrying LPS type L3 had been successful in protecting against challenge from L3 isolates it might have driven the emergence of the closely related clade 2, against which the vaccine was ineffective. The genomic results also revealed a 14 bp deletion in the galactosyltransferase gene gatG in LPS type L3 isolates, which would result in producing a semi-truncated LPS in those isolates. In conclusion, our study clearly demonstrates the advantages of genomic analysis over the conventional PCR-based approaches in providing clear insights in terms of linkage of isolate within and between outbreaks. More importantly, it provides more detailed information than the multiplex PCR on the possible structure of outer LPS, which is very important in the case of strain selection for killed autogenous vaccines.

The emergence of polymyxin resistance in carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria is a critical threat to human health, and alternative treatment strategies are urgently required. We investigated the ability of the hydroxyquinoline analog ionophore PBT2 to restore antibiotic sensitivity in polymyxin-resistant, ESBL-producing, carbapenem-resistant Gram-negative human pathogens. PBT2 resensitized Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa to last-resort polymyxin class antibiotics, including the less toxic next-generation polymyxin derivative FADDI-287, in vitro. We were unable to select for mutants resistant to PBT2 + FADDI-287 in polymyxin-resistant E. coli containing a plasmid-borne mcr-1 gene or K. pneumoniae carrying a chromosomal mgrB mutation. Using a highly invasive K. pneumoniae strain engineered for polymyxin resistance through mgrB mutation, we successfully demonstrated the efficacy of PBT2 + polymyxin (colistin or FADDI-287) for the treatment of Gram-negative sepsis in immunocompetent mice. In comparison to polymyxin alone, the combination of PBT2 + polymyxin improved survival and reduced bacterial dissemination to the lungs and spleen of infected mice. These data present a treatment modality to break antibiotic resistance in high-priority polymyxin-resistant Gram-negative pathogens.

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak., Antibiotic-resistant bacteria are an urgent threat to human health. Here, Roberts et al. characterise and monitor an ongoing hospital outbreak of carbapenemase-producing Enterobacter hormaechei by integrating several technologies for whole-genome sequencing and shotgun metagenomics.

Fish mortality caused by Streptococcus iniae is a major economic problem in fish aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by S. iniae through handling of contaminated fish. In this study, we present the complete genome sequence of S. iniae strain QMA0248, isolated from farmed barramundi in South Australia. The 2.12 Mb genome of S. iniae QMA0248 carries a 32 Kb prophage, a 12 Kb genomic island, and 92 discrete insertion sequence (IS) elements. These include 9 novel IS types that belong mostly to the IS3 family. Comparative and phylogenetic analysis between S. iniae QMA0248 and publicly available complete S. iniae genomes revealed discrepancies that are likely due to misassembly in the genomes of isolates ISET0901 and ISNO. We also determined by long-range PCR that a tandem duplication of an rRNA region in the PacBio assembly of QMA0248 was an assembly error. A similar rRNA duplication in the PacBio genome of S. iniae 89353 may also be a misassembly. Our study not only highlights assembly problems in existing genomes, but provides a high quality reference genome for S. iniae QMA0248, including manually curated mobile genetic elements, that will assist future S. iniae comparative genomic and evolutionary studies.