HAIviz: an interactive dashboard for visualising and integrating healthcare-associated genomic epidemiological data.

Existing tools for phylogeographic and epidemiological visualisation primarily provide a macro-geographic view of epidemic and pandemic transmission events but offer little support for detailed investigation of outbreaks in healthcare settings. Here, we present HAIviz, an interactive web-based application designed for integrating and visualising genomic epidemiological information to improve the tracking of healthcare-associated infections (HAIs). HAIviz displays and links the outbreak timeline, building map, phylogenetic tree, patient bed movements, and transmission network on a single interactive dashboard. HAIviz has been developed for bacterial outbreak investigations but can be utilised for general epidemiological investigations focused on built environments for which visualisation to customised maps is required. This paper describes and demonstrates the application of HAIviz for HAI outbreak investigations.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data.

BACKGROUND: DNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. METHODS: In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan 14, 2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. FINDINGS: We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), and linezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. INTERPRETATION: To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. FUNDING: Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

Long-read sequencing reveals genomic diversity and associated plasmid movement of carbapenemase-producing bacteria in a UK hospital over 6 years.

Healthcare-associated infections (HCAIs) affect the most vulnerable people in society and are increasingly difficult to treat in the face of mounting antimicrobial resistance (AMR). Routine surveillance represents an effective way of understanding the circulation and burden of bacterial resistance and transmission in hospital settings. Here, we used whole-genome sequencing (WGS) to retrospectively analyse carbapenemase-producing Gram-negative bacteria from a single hospital in the UK over 6 years (n=165). We found that the vast majority of isolates were either hospital-onset (HAI) or HCAI. Most carbapenemase-producing organisms were carriage isolates, with 71 % isolated from screening (rectal) swabs. Using WGS, we identified 15 species, the most common being Escherichia coli and Klebsiella pneumoniae. Only one significant clonal outbreak occurred during the study period and involved a sequence type (ST)78 K. pneumoniae carrying bla NDM-1 on an IncFIB/IncHI1B plasmid. Contextualization with public data revealed little evidence of this ST outside of the study hospital, warranting ongoing surveillance. Carbapenemase genes were found on plasmids in 86 % of isolates, the most common types being bla NDM- and bla OXA-type alleles. Using long-read sequencing, we determined that approximately 30 % of isolates with carbapenemase genes on plasmids had acquired them via horizontal transmission. Overall, a national framework to collate more contextual genomic data, particularly for plasmids and resistant bacteria in the community, is needed to better understand how carbapenemase genes are transmitted in the UK.

Genomic characterisation of multidrug-resistant Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii in two intensive care units in Hanoi, Viet Nam: a prospective observational cohort study.

BACKGROUND: Viet Nam has high rates of antimicrobial resistance (AMR) but little capacity for genomic surveillance. This study used whole genome sequencing to examine the prevalence and transmission of three key AMR pathogens in two intensive care units (ICUs) in Hanoi, Viet Nam. METHODS: A prospective surveillance study of all adults admitted to ICUs at the National Hospital for Tropical Diseases and Bach Mai Hospital was done between June 19, 2017, and Jan 16, 2018. Clinical and environmental samples were cultured on selective media, characterised with MALDI TOF mass spectrometry, and sequenced with Illumina. Phylogenies based on the de-novo assemblies (SPAdes) were constructed with MAFFT (PARsnp), Gubbins, and RAxML. Resistance genes were detected with Abricate against the US National Center for Biotechnology Information database. FINDINGS: 3153 Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii isolates from 369 patients were analysed. Phylogenetic analysis revealed predominant lineages within A baumannii (global clone 2, sequence types ST2 and ST571) and K pneumoniae (ST15, ST16, ST656, ST11, and ST147) isolates. Isolation from stool was most common with E coli (87·0%) followed by K pneumoniae (62·5%). Of the E coli, 85·0% carried a blaCTX-M variant, while 81·8% of K pneumoniae isolates carried blaNDM (54·4%), or blaKPC (45·1%), or both. Transmission analysis with single nucleotide polymorphisms identified 167 clusters involving 251 (68%) of 369 patients, in some cases involving patients from both ICUs. There were no clear differences between the lineages or AMR genes recovered between the two ICUs. INTERPRETATION: This study represents the largest prospective surveillance study of key AMR pathogens in Vietnamese ICUs. Clusters of closely related isolates in patients across both ICUs suggests recent transmission before ICU admission in other health-care settings or in the community. FUNDING: UK Medical Research Council Newton Fund, Viet Nam Ministry of Science and Technology, Wellcome Trust, Academy of Medical Sciences, Health Foundation, and UK National Institute for Health and Care Research Cambridge Biomedical Research Centre.

Differential Afa/Dr Fimbriae Expression in the Multidrug-Resistant Escherichia coli ST131 Clone.

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.

Pandora: nucleotide-resolution bacterial pan-genomics with reference graphs.

We present pandora, a novel pan-genome graph structure and algorithms for identifying variants across the full bacterial pan-genome. As much bacterial adaptability hinges on the accessory genome, methods which analyze SNPs in just the core genome have unsatisfactory limitations. Pandora approximates a sequenced genome as a recombinant of references, detects novel variation and pan-genotypes multiple samples. Using a reference graph of 578 Escherichia coli genomes, we compare 20 diverse isolates. Pandora recovers more rare SNPs than single-reference-based tools, is significantly better than picking the closest RefSeq reference, and provides a stable framework for analyzing diverse samples without reference bias.

MicroPIPE: validating an end-to-end workflow for high-quality complete bacterial genome construction.

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 .

Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care.

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.

Characterization of DtrJ as an IncC plasmid conjugative DNA transfer component.

Incompatibility group C (IncC) plasmids are large (50-400 kb), broad host range plasmids that drive the spread of genes conferring resistance to all classes of antibiotics, most notably the blaNDM gene that confers resistance to last-line carbapenems and the mcr-3 gene that confers resistance to colistin. Several recent studies have improved our understanding of the basic biological mechanisms driving the success of IncC, in particular the identification of multiple novel IncC conjugation genes by transposon directed insertion-site sequencing. Here, one of these genes, dtrJ, was examined in further detail. The dtrJ gene is located in the DNA transfer locus on the IncC backbone, and quantitative reverse-transcriptase PCR analysis revealed it is transcribed in the same operon as the DNA transfer genes traI and traD (encoding the relaxase and coupling protein, respectively) and activated by the AcaDC regulatory complex. We confirmed that DtrJ is not required for pilus biogenesis or mate pair formation. Instead, DtrJ localizes to the membrane, where it interacts with the coupling protein TraD and functions as an IncC DNA transfer protein. Overall, this work has defined the role of DtrJ in DNA transfer of IncC plasmids during conjugation.

Cost-effectiveness analysis of whole-genome sequencing during an outbreak of carbapenem-resistant Acinetobacter baumannii.

Background: Whole-genome sequencing (WGS) shotgun metagenomics (metagenomics) attempts to sequence the entire genetic content straight from the sample. Diagnostic advantages lie in the ability to detect unsuspected, uncultivatable, or very slow-growing organisms. Objective: To evaluate the clinical and economic effects of using WGS and metagenomics for outbreak management in a large metropolitan hospital. Design: Cost-effectiveness study. Setting: Intensive care unit and burn unit of large metropolitan hospital. Patients: Simulated intensive care unit and burn unit patients. Methods: We built a complex simulation model to estimate pathogen transmission, associated hospital costs, and quality-adjusted life years (QALYs) during a 32-month outbreak of carbapenem-resistant Acinetobacter baumannii (CRAB). Model parameters were determined using microbiology surveillance data, genome sequencing results, hospital admission databases, and local clinical knowledge. The model was calibrated to the actual pathogen spread within the intensive care unit and burn unit (scenario 1) and compared with early use of WGS (scenario 2) and early use of WGS and metagenomics (scenario 3) to determine their respective cost-effectiveness. Sensitivity analyses were performed to address model uncertainty. Results: On average compared with scenario 1, scenario 2 resulted in 14 fewer patients with CRAB, 59 additional QALYs, and $75,099 cost savings. Scenario 3, compared with scenario 1, resulted in 18 fewer patients with CRAB, 74 additional QALYs, and $93,822 in hospital cost savings. The likelihoods that scenario 2 and scenario 3 were cost-effective were 57% and 60%, respectively. Conclusions: The use of WGS and metagenomics in infection control processes were predicted to produce favorable economic and clinical outcomes.

Genomic Investigation Reveals Contaminated Detergent as the Source of an Extended-Spectrum-ß-Lactamase-Producing Klebsiella michiganensis Outbreak in a Neonatal Unit.

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-ß-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.

Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei.

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.

Genomic analysis of carbapenemase-producing Enterobacteriaceae in Queensland reveals widespread transmission of blaIMP-4 on an IncHI2 plasmid.

Carbapenemase-producing Enterobacteriaceae (CPE) are an increasingly common cause of healthcare-associated infections and may occasionally be identified in patients without extensive healthcare exposure. blaIMP-4 is the most frequently detected carbapenemase gene in Enterobacteriaceae within Australia, but little is known about the mechanisms behind its persistence. Here we used whole genome sequencing (WGS) to investigate the molecular epidemiology of blaIMP-4 in Queensland, Australia. In total, 107 CPE were collected between 2014 and 2017 and sent for WGS on an Illumina NextSeq500. Resistance genes and plasmid types were detected using a combination of read mapping and nucleotide comparison of de novo assemblies. Six isolates were additionally sequenced using Oxford Nanopore MinION to generate long-reads and fully characterize the context of the blaIMP-4 gene. Of 107 CPE, 93 carried the blaIMP-4 gene; 74/107 also carried an IncHI2 plasmid, suggesting carriage of the blaIMP-4 gene on an IncHI2 plasmid. Comparison of these isolates to a previously characterized IncHI2 plasmid pMS7884A (isolated from an Enterobacter hormaechei strain in Brisbane) suggested that all isolates carried a similar plasmid. Five of six representative isolates sequenced using Nanopore long-read technology carried IncHI2 plasmids harbouring the blaIMP-4 gene. While the vast majority of isolates represented E. hormaechei, several other species were also found to carry the IncHI2 plasmid, including Klebsiella species, Escherichia coli and Citrobacter species. Several clonal groups of E. hormaechei were also identified, suggesting that persistence of blaIMP-4 is driven by both presence on a common plasmid and clonal spread of certain E. hormaechei lineages.

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

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.

Detection of Epidemic Scarlet Fever Group A Streptococcus in Australia.

Sentinel hospital surveillance was instituted in Australia to detect the presence of pandemic group A Streptococcus strains causing scarlet fever. Genomic and phylogenetic analyses indicated the presence of an Australian GAS emm12 scarlet fever isolate related to United Kingdom outbreak strains. National surveillance to monitor this pandemic is recommended.

Whole genome analysis of cephalosporin-resistant Escherichia coli from bloodstream infections in Australia, New Zealand and Singapore: high prevalence of CMY-2 producers and ST131 carrying blaCTX-M-15 and blaCTX-M-27.

Objectives: To characterize MDR Escherichia coli from bloodstream infections (BSIs) in Australia, New Zealand and Singapore. Methods: We collected third-generation cephalosporin-resistant (3GC-R) E. coli from blood cultures in patients enrolled in a randomized controlled trial from February 2014 to August 2015. WGS was used to characterize antibiotic resistance genes, MLST, plasmids and phylogenetic relationships. Antibiotic susceptibility was determined using disc diffusion and Etest. Results: A total of 70 3GC-R E. coli were included, of which the majority were ST131 (61.4%). BSI was most frequently from a urinary source (69.6%), community associated (62.9%) and in older patients (median age 71 years). The median Pitt score was 1 and ICU admission was infrequent (3.1%). ST131 possessed more acquired resistance genes than non-ST131 (P = 0.003). Clade C1/C2 ST131 predominated (30.2% and 53.5% of ST131, respectively) and these were all ciprofloxacin resistant. All clade A ST131 (n = 6) were community associated. The predominant ESBL types were blaCTX-M (80.0%) and were strongly associated with ST131 (95% carried blaCTX-M), with the majority blaCTX-M-15. Clade C1 was associated with blaCTX-M-14 and blaCTX-M-27, whereas blaCTX-M-15 predominated in clade C2. Plasmid-mediated AmpC genes (mainly blaCMY-2) were frequent (17.1%) but were more common in non-ST131 (P < 0.001) isolates from Singapore and Brisbane. Two strains carried both blaCMY-2 and blaCTX-M. The majority of plasmid replicon types were IncF. Conclusions: In a prospective collection of 3GC-R E. coli causing BSI, community-associated Clade C1/C2 ST131 predominate in association with blaCTX-M ESBLs, although a significant proportion of non-ST131 strains carried blaCMY-2.

Comprehensive analysis of type 1 fimbriae regulation in fimB-null strains from the multidrug resistant Escherichia coli ST131 clone.

Uropathogenic Escherichia coli (UPEC) of sequence type 131 (ST131) are a pandemic multidrug resistant clone associated with urinary tract and bloodstream infections. Type 1 fimbriae, a major UPEC virulence factor, are essential for ST131 bladder colonization. The globally dominant sub-lineage of ST131 strains, clade C/H30-R, possess an ISEc55 insertion in the fimB gene that controls phase-variable type 1 fimbriae expression via the invertible fimS promoter. We report that inactivation of fimB in these strains causes altered regulation of type 1 fimbriae expression. Using a novel read-mapping approach based on Illumina sequencing, we demonstrate that 'off' to 'on' fimS inversion is reduced in these strains and controlled by recombinases encoded by the fimE and fimX genes. Unlike typical UPEC strains, the nucleoid-associated H-NS protein does not strongly repress fimE transcription in clade C ST131 strains. Using a genetic screen to identify novel regulators of fimE and fimX in the clade C ST131 strain EC958, we defined a new role for the guaB gene in the regulation of type 1 fimbriae and in colonisation of the mouse bladder. Our results provide a comprehensive analysis of type 1 fimbriae regulation in ST131, and highlight important differences in its control compared to non-ST131 UPEC.

Sequential Acquisition of Virulence and Fluoroquinolone Resistance Has Shaped the Evolution of Escherichia coli ST131.

UNLABELLED: Escherichia coli ST131 is the most frequently isolated fluoroquinolone-resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Although originally identified through its association with the CTX-M-15 extended-spectrum ß-lactamase resistance gene, global genomic epidemiology studies have failed to resolve the geographical and temporal origin of the ST131 ancestor. Here, we developed a framework for the reanalysis of publically available genomes from different countries and used this data set to reconstruct the evolutionary steps that led to the emergence of FQR ST131. Using Bayesian estimation, we show that point mutations in chromosomal genes that confer FQR coincide with the first clinical use of fluoroquinolone in 1986 and illustrate the impact of this pivotal event on the rapid population expansion of ST131 worldwide from an apparent origin in North America. Furthermore, we identify virulence factor acquisition events that predate the development of FQR, suggesting that the gain of virulence-associated genes followed by the tandem development of antibiotic resistance primed the successful global dissemination of ST131. IMPORTANCE: Escherichia coli sequence type 131 (ST131) is a recently emerged and globally disseminated multidrug-resistant clone frequently associated with human urinary tract and bloodstream infections. In this study, we have used two large publically available genomic data sets to define a number of critical steps in the evolution of this important pathogen. We show that resistance to fluoroquinolones, a class of broad-spectrum antibiotic used extensively in human medicine and veterinary practice, developed in ST131 soon after the introduction of these antibiotics in the United States, most likely in North America. We also mapped the acquisition of several fitness and virulence determinants by ST131 and demonstrate these events occurred prior to the development of fluoroquinolone resistance. Thus, ST131 has emerged by stealth, first acquiring genes associated with an increased capacity to cause human infection, and then gaining a resistance armory that has driven its massive population expansion across the globe.

Third-generation cephalosporin resistance conferred by a chromosomally encoded blaCMY-23 gene in the Escherichia coli ST131 reference strain EC958.

OBJECTIVES: Escherichia coli ST131 is a globally disseminated MDR clone originally identified due to its association with the blaCTX-M-15 gene encoding an ESBL. It is thus assumed that blaCTX-M-15 is the major determinant for resistance to ß-lactam antibiotics in this clone. The complete sequence of EC958, a reference strain for E. coli ST131, revealed that it contains a chromosomally located blaCMY-23 gene with an upstream ISEcp1 element as well as several additional plasmid-encoded ß-lactamase genes. Here, we examined the genetic context of the blaCMY-23 element in EC958 and other E. coli ST131 strains and investigated the contribution of blaCMY-23 to EC958 resistance to a range of ß-lactam antibiotics. METHODS: The genetic context of blaCMY-23 and its associated mobile elements was determined by PCR and sequencing. Antibiotic susceptibility testing was performed using Etests. The activity of the blaCMY-23 promoter was assessed using lacZ reporter assays. Mutations were generated using λ-Red-recombination. RESULTS: The genetic structure of the ISEcp1-IS5-blaCMY-23 mobile element was determined and localized within the betU gene on the chromosome of EC958 and five other E. coli ST131 strains. The transcription of blaCMY-23, driven by a previously defined promoter within ISEcp1, was significantly higher than other ß-lactamase genes and could be induced by cefotaxime. Deletion of the blaCMY-23 gene resulted in enhanced susceptibility to cefoxitin, cefotaxime and ceftazidime. CONCLUSIONS: This is the first known report to demonstrate the chromosomal location of blaCMY-23 in E. coli ST131. In EC958, CMY-23 plays a major role in resistance to third-generation cephalosporins and cephamycins.