Pediatric blood cultures-turning up the volume: a before and after intervention study.

The major determinant of blood culture (BC) diagnostic performance is blood volume, and pediatric sample volumes are frequently low. We aimed to assess BC volumes in our institution, design an intervention to increase volumes, and assess its impact. All pediatric BCs submitted over a 7-month period to the microbiology laboratory in a university hospital (including emergency department, pediatric ward, and neonatal unit) were included. A pre-intervention period assessed current practice. A multi-faceted intervention (education, guideline introduction, active feedback strategies) was collaboratively designed by all stakeholders. Impact was assessed in a post-intervention period. The main outcome measures included the percentage of samples adequately filled using three measures of sample adequacy (1) manufacturer-recommended minimum validated volume-> 0.5 ml, (2) manufacturer-recommended optimal minimum volume-> 1.0 ml, (3) newly introduced age-specific recommendations. Three hundred ninety-eight pre-intervention and 388 post-intervention samples were included. Initial volumes were low but increased significantly post-intervention (median 0.77 ml vs. 1.52 ml), with multivariable regression analysis estimating volumes increased 89% post-intervention. There were significant increases in all measures of volume adequacy, including an increase in age-appropriate filling (20.4-53.1%), with less improvement in those aged > 3 years. Overall, 68.4% of pathogens were from adequately filled cultures, while 76% of contaminants were from inadequately filled cultures. A pathogen was detected in a higher proportion of adequately filled than inadequately filled cultures (9.4% vs. 2.2%, p < 0.001).  Conclusion: Blood volume impacts BC sensitivity, with lower volumes yielding fewer pathogens and more contaminants. Focused intervention can significantly improve volumes to improve diagnostic performance. What is Known: • Blood volume is the major determinant of blood culture positivity, and yet pediatric blood culture volumes are frequently low, resulting in missed pathogens and increased contamination. What is New: • Adequately filled (for age) blood cultures have a pathogen detection rate three times higher than inadequately filled blood cultures. • This interventional study shows that collaboratively designed multi-modal interventions including focus on accurate volume measurement can lead to significant increases in blood volumes and improve blood culture diagnostic performance.

Paediatric Escherichia coli urinary tract infection: susceptibility trends and clinical management-a retrospective analysis of a 10-year period.

BACKGROUND: Escherichia coli is the predominant urinary pathogen in children. Irish and international studies have demonstrated increasing antimicrobial resistance (AMR) to antibiotics such as co-amoxiclav. AIMS: We aimed to (1) examine the AMR patterns of paediatric urinary E. coli isolates, from both hospital and community sources, over a 10-year period; (2) assess the effectiveness of Children's Health Ireland (CHI) antimicrobial guidance given local susceptibility data; and (3) review the clinical management of an admitted patient sub-set over a 6-year period. METHODS: Pure growth of urinary E. coli from patients aged ≤ 14 from 2012 to 2021 were analysed for AMR. Differences in susceptibility rates were assessed. A retrospective chart review conducted on inpatients aged ≥ 2 months to ≤ 14 years, 2016-2021. RESULTS: E. coli accounted for 70.8% of likely significant positive pure growth cultures (9314 isolates). Susceptibility to co-amoxiclav significantly increased over time, from 66.7% to 80.4% (2016-2021, p < 0.001). Nitrofurantoin and cefalexin had significantly higher susceptibility rates than trimethoprim (< 70% annually). 85.1% of isolates were susceptible to the combination of co-amoxiclav and gentamicin, recommended for those > 2months and systemically unwell. The additional gain in empiric susceptibility provided by gentamicin above that provided by co-amoxiclav alone has fallen from 16.4% to 6.7% (2016-2021). The 222 clinical cases reviewed showed improved antimicrobial guideline compliance over time. CONCLUSIONS: This study provides important regional AMR data. Co-amoxiclav susceptibility increased significantly over time, contrasting with previous studies. This was temporally associated with stewardship measures reducing co-amoxiclav prescribing. Decreasing utility of gentamicin supports recent CHI guideline updates reducing gentamicin use.

Longitudinal carriage of antimicrobial resistant Enterobacterales in healthy individuals in Ireland - Assessing the impact of recreational water use on duration of carriage.

The increasing prevalence of extended-spectrum beta-lactamase (ESBL) producing Enterobacterales (ESBL-PE) and carbapenemase-producing Enterobacterales (CPE) is a major public health concern worldwide. Despite the associated risk of infection from gut colonisation with a resistant Enterobacterales, the incidence and duration of carriage in healthy individuals is poorly studied. This "persistence study" is the first in Ireland to assess the longitudinal carriage of ESBL-PE and CPE in healthy individuals. A cohort of 45 participants, 22 of whom were colonised with ESBL-PE, was recruited from a recently completed point prevalence study that investigated colonisation in recreational water users (WU) versus controls. Six bi-monthly faecal samples per participant were analysed for CPE and ESBL-PE over one year and the relationship between persistent colonisation and exposure to natural waters was investigated. For 11 of 45 participants (24.4 %) ESBL-E. coli (ESBL-EC) was detected in at least one sample. Genomic analysis revealed that six participants harboured the same ESBL-EC strains as identified in the preceding study. ESBL-EC persisted in the gut for a median duration of 10.3 months (range 4-23 months), consistent with previous research. Five participants (11.1 %) carried ESBL-EC for the entire study year. The carbapenemase gene blaIMI-2 was detected once. Colonisation was higher in water users during the non-bathing season (n = 10, November 2021-April 2022), than during the bathing season (n = 5, May 2022-September 2022) [relative risk 1.99 (95 % CI 0.34-11.71)]. However, overall WU were less likely to be colonised with ESBL-EC than controls (19 % vs 25 % respectively, RR 0.76, CI 0.24-2.34). Further research is warranted to better understand the factors influencing the persistence of gut colonisation with ESBL-EC and CPE and to what extent bathing water quality impacts colonisation for those regularly exposed.

A global genomic analysis of Salmonella Concord reveals lineages with high antimicrobial resistance in Ethiopia.

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance.

Medical Astro-Microbiology: Current Role and Future Challenges.

The second and third decades of the twenty-first century are marked by a flourishing of space technology which may soon realise human aspirations of a permanent multiplanetary presence. The prevention, control and management of infection with microbial pathogens is likely to play a key role in how successful human space aspirations will become. This review considers the emerging field of medical astro-microbiology. It examines the current evidence regarding the risk of infection during spaceflight via host susceptibility, alterations to the host's microbiome as well as exposure to other crew members and spacecraft's microbiomes. It also considers the relevance of the hygiene hypothesis in this regard. It then reviews the current evidence related to infection risk associated with microbial adaptability in spaceflight conditions. There is a particular focus on the International Space Station (ISS), as one of the only two  crewed objects in low Earth orbit. It discusses the effects of spaceflight related stressors on viruses and the infection risks associated with latent viral reactivation and increased viral shedding during spaceflight. It then examines the effects of the same stressors on bacteria, particularly in relation to changes in virulence and drug resistance. It also considers our current understanding of fungal adaptability in spaceflight. The global public health and environmental risks associated with a possible re-introduction to Earth of invasive species are also briefly discussed. Finally, this review examines the largely unknown microbiology and infection implications of celestial body habitation with an emphasis placed on Mars. Overall, this review summarises much of our current understanding of medical astro-microbiology and identifies significant knowledge gaps.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Assessing the impact of recreational water use on carriage of antimicrobial resistant organisms.

Understanding the role of exposure to natural recreational waters in the acquisition and transmission of antimicrobial resistance (AMR) is an area of increasing interest. A point prevalence study was carried out in the island of Ireland to determine the prevalence of colonisation with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE) in recreational water users (WU) and matched controls. A total of 411 adult participants (199 WU, 212 controls) submitted at least one faecal sample between September 2020 - October 2021. In total, 80 Enterobacterales were isolated from 73 participants. ESBL-PE were detected in 29 (7.1 %) participants (7 WU, 22 controls), and CRE were detected in nine (2.2 %) participants (4 WU, 5 controls). No carbapenemase-producing Enterobacterales (CPE) were detected. WU were significantly less likely to harbour ESBL-PE than controls (risk ratio = 0.34, 95 % CI 0.148 to 0.776, χ2 7.37, p = 0.007). This study demonstrates the occurrence of ESBL-PE and CRE in healthy participants in Ireland. Recreational exposure to bathing water in Ireland was associated with a decreased prevalence of colonisation with ESBL-PE and CRE.

Dissemination of carbapenemase-producing Enterobacterales in Ireland from 2012 to 2017: a retrospective genomic surveillance study.

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within-host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species (Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter hormaechei and Citrobacter freundii) were individually examined for evidence of transmission. Antimicrobial resistance trends over time were also assessed. We identified 352 putative transmission events in six species including widespread and frequent transmissions in three species. We detected putative outbreaks in 4/6 species with three hospitals experiencing prolonged outbreaks. The bla OXA-48 gene was the main cause of carbapenem resistance in Ireland in almost all species. An expansion in the number of sequence types carrying bla OXA-48 was an additional cause of the increasing prevalence of carbapenemase-producing K. pneumoniae and E. coli.

First reported detection of the mobile colistin resistance genes, mcr-8 and mcr-9, in the Irish environment.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n = 2), healthcare facility wastewater (n = 2), wastewater treatment plant influent (n = 1) and integrated constructed wetland influent (piggery farm waste) (n = 1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 ± 4.1 (10-61), including the carbapenemases, blaOXA-48 (n = 2) and blaNDM-1 (n = 1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

COSUTI: A Core Outcome Set (COS) for Interventions for the Treatment of Uncomplicated Urinary Tract Infection (UTI) in Adults.

Background: Uncomplicated urinary tract infections (UTIs) are among the most common presentations of bacterial infections in the outpatient setting. The variation of outcomes reported in trials to assess the most effective treatment interventions for uncomplicated UTIs has meant that comparing and synthesising the outcomes across trials is challenging and limits the reliability of evidence which would otherwise inform healthcare decisions. Objective: Develop a Core Outcome Set (COS) for interventions for the treatment of uncomplicated UTIs in otherwise healthy adults. Methods: The COS development consisted of three phases: (1) A systematic review to identify outcomes reported in randomised trials and systematic reviews of randomised trials comparing the effectiveness of any interventions for the treatment of uncomplicated UTI in otherwise healthy adults; (2) Outcomes identified in the systematic review were prioritised in an online 3-round modified Delphi survey with healthcare practitioners (n = 68), researchers (n = 5), and people who have experienced or cared for someone experiencing a UTI (n = 180); (3) An online consensus meeting to determine the final COS with healthcare practitioners and policymakers (n = 9), researchers (n = 4), and people who have experienced or cared for someone experiencing a UTI (n = 7). Results: We identified a large number of outcomes. Through the use of robust consensus methods, those outcomes were reduced to a core set of six outcomes that should, at a minimum, be measured and reported in randomised trials and systematic reviews of interventions treating uncomplicated UTIs in adults.

A geographic cluster of healthcare-associated carbapenemase-producing hypervirulent Klebsiella pneumoniae sequence type 23.

Hypervirulent Klebsiella pneumoniae has typically been associated with invasive, community-associated infections. This study describes the molecular, epidemiological and clinical characteristics of a cluster of carbapenemase-producing hypervirulent K. pneumoniae in the South-East of Ireland. It highlights the increasing risk that hypervirulent K. pneumoniae poses to healthcare and residential care populations. A retrospective analysis of sequences on K. pneumoniae isolates in the K. pneumoniae database of the National Carbapenemase-Producing Enterobacterales Reference Laboratory Service was performed to identify cases of hypervirulent K. pneumoniae from one hospital network. Hypervirulence scores were assigned based on the presence of recognised hypervirulence genes. A retrospective review of patient records was carried out for all confirmed cases of hypervirulent K. pneumoniae identified and clinical, epidemiological and molecular characteristics described. Twenty-eight cases of hypervirulent OXA-48 producing K. pneumoniae were identified over a 2-year period. All isolates were sequence-type 23 with a hypervirulence score of 5. All isolates carried the blaOXA-48 carbapenemase gene. All cases had a record of current or recent hospitalisation or residence in a long-term residential care facility. This study describes extensive dissemination of hypervirulent K. pneumoniae within healthcare facilities and an ongoing outbreak in our region. It shows the convergence of hypervirulence and antibiotic resistance determinants. Healthcare facilities need to consider their infection prevention, control and surveillance strategies to monitor and prevent further dissemination among a vulnerable population. Diagnostic laboratories need to ensure they have the ability and capacity for testing. Readily deployed laboratory methods for detection of hypervirulence are required.

A Longitudinal Survey of Antibiotic-Resistant Enterobacterales in the Irish Environment, 2019-2020.

The natural environment represents a complex reservoir of antibiotic-resistant bacteria as a consequence of different wastewater discharges including anthropogenic and agricultural. Therefore, the aim of this study was to examine sewage and waters across Ireland for the presence of antibiotic-resistant Enterobacterales. Samples were collected from the West, East and South of Ireland. Two periods of sampling took place between July 2019 and November 2020, during which 118 water (30 L) and 36 sewage samples (200 mL) were collected. Waters were filtered using the CapE method, followed by enrichment and culturing. Sewage samples were directly cultured on selective agars. Isolates were identified by MALDI-TOF and antibiotic susceptibility testing was performed in accordance with EUCAST criteria. Selected isolates were examined for blaCTX-M, blaVIM, blaIMP, blaOXA-48, blaNDM, and blaKPC by real time PCR and whole genome sequencing (n = 146). A total of 419 Enterobacterales (348 water, 71 sewage) were isolated from all samples. Hospital sewage isolates displayed the highest percentage resistance to many beta-lactam and aminoglycoside antibiotics. Extended-spectrum beta-lactamase-producers were identified in 78% of water and 50% of sewage samples. One or more carbapenemase-producing Enterobacterales were identified at 23 individual sampling sites (18 water, 5 sewage). This included the detection of blaOXA-48 (n = 18), blaNDM (n = 14), blaKPC (n = 4) and blaOXA-484 (n = 1). All NDM-producing isolates harbored the ble-MBL bleomycin resistance gene. Commonly detected sequence types included Klebsiella ST323, ST17, and ST405 as well as E. coli ST131, ST38 and ST10. Core genome MLST comparisons detected identical E. coli isolates from wastewater treatment plant (WWTP) influent and nursing home sewage, and the surrounding waters. Similarly, one Klebsiella pneumoniae isolated from WWTP influent and the surrounding estuarine water were identical. These results highlight the need for regular monitoring of the aquatic environment for the presence of antibiotic-resistant organisms to adequately inform public health policies.

A systematic review of the outcomes reported in the treatment of uncomplicated urinary tract infection clinical trials.

Background: Uncomplicated urinary tract infections (UTIs) are amongst the most frequent infections presenting in the outpatient setting. A growing number of clinical trials are assessing the most effective treatment interventions for uncomplicated UTI. Due to the heterogeneity of the outcomes reported in these trials, however, comparing these outcomes is challenging. Objectives: Identify the core outcomes that have been reported in trials and systematic reviews of interventions treating uncomplicated UTI in adults. Methods: We conducted a systematic search for core outcomes used to evaluate treatments of UTIs. We searched the Cochrane Database of Systematic Reviews, PubMed and Embase. One researcher independently screened each article for inclusion, and the Core Outcome Set for treatment of Urinary Tract Infections (COSUTI) team acted as second reviewers. All included articles were screened by two reviewers. All outcomes were extracted verbatim, and similar outcomes were grouped into domains and subdomains. Results: In total, 334 outcomes were reported across 41 papers, the average number of outcomes reported being 8. Outcomes were categorized across 18 domains, the majority of which were related to clinical cure outcomes. Many outcomes varied in the timepoints within which the outcome was measured and reported. Conclusions: Comparing the outcomes of trials investigating uncomplicated UTI treatment remains challenging due to the difference in outcomes currently reported. Consistency of reporting of outcomes would be improved by developing a minimum number of consistent outcomes that should be reported in all trials.

Presentation, Treatment, and Natural Course of Severe Symptoms of Urinary Tract Infections Measured by a Smartphone App: Observational and Feasibility Study.

BACKGROUND: Urinary tract infections (UTIs) are one of the most common conditions in women. Current information on the presentation, management, and natural course of the infection is based on paper diaries filled out and subsequently posted by patients. OBJECTIVE: The aim of this study is to explore the feasibility of a smartphone app to assess the natural course and management of UTIs. METHODS: A smartphone app was developed to collect data from study participants presenting with symptoms of UTI in general practice. After initial demographic and treatment information, symptom severity was recorded by the patient after a reminder on their smartphone, which occurred twice daily for a period of 7 days or until symptom resolution. RESULTS: A total of 181 women aged 18-76 years downloaded the smartphone app. The duration of symptoms was determined from the results of 178 participants. All patients submitted a urine sample, most patients were prescribed an antibiotic (163/181, 90.1%), and 38.7% (70/181) of the patients had a positive culture. Moderately bad or worse symptoms lasted a mean of 3.8 (SD 3.2; median 4) days, and 70.2% (125/178) of the patients indicated that they were cured on day 4 after consultation. This compares with other research assessing symptom duration and management of UTIs using paper diaries. Patients were very positive about the usability of the smartphone app and often found the reminders supportive. On the basis of the feedback and the analysis of the data, some suggestions for improvement were made. CONCLUSIONS: Smartphone diaries for symptom scores over the course of infections are an efficient and acceptable means of collecting data in research.

Listeria infection in young infants: results from a national surveillance study in the UK and Ireland.

OBJECTIVES: To describe the epidemiology, age at infection, clinical characteristics and outcome of listeria infection in young infants to inform management and empiric antibiotic choice in young infants. DESIGN: Prospective 2-year surveillance of Listeria monocytogenes infection in young infants detected through the British Paediatric Surveillance Unit 'orange card' system and triangulated with the public health laboratories. SETTING: National population study (England, Wales, Scotland and the Ireland) PATIENTS: All infants under 90 days with proven or probable invasive listeriosis MAIN OUTCOME MEASURES: Incidence, mortality, age of infection, clinical characteristics and outcome RESULTS: During a 2-year period (2017-2019), 27 cases of listeriosis in infants <90 days of age were reported. The incidence of listeriosis in this study was 1.8 per 100 000 live births with 7% mortality (2/27). Nearly all cases presented within the first 24 hours of life (26/27). The majority (20/27, 74%) were born preterm and 16/24 (67%) were born to women from ethnic minority backgrounds. CONCLUSIONS: Invasive listeriosis in young infants in the UK and Ireland is rare and presents early in the neonatal period. National guidelines that recommend the use of amoxicillin as part of empiric regimes for sepsis and meningitis in infants over 1 month of age should be modified.

A Point Prevalence Survey of Antibiotic Resistance in the Irish Environment, 2018-2019.

Water bodies worldwide have proven to be vast reservoirs of clinically significant antibiotic resistant organisms. Contamination of waters by anthropogenic discharges is a significant contributor to the widespread dissemination of antibiotic resistance. The aim of this research was to investigate multiple different anthropogenic sources on a national scale for the role they play in the environmental propagation of antibiotic resistance. A total of 39 water and 25 sewage samples were collected across four local authority areas in the West, East and South of Ireland. In total, 211 Enterobacterales were isolated (139 water, 72 sewage) and characterised. A subset of isolates (n=60) were chosen for whole genome sequencing. Direct comparisons of the water versus sewage isolate collections revealed a higher percentage of sewage isolates displayed resistance to cefoxitin (46%) and ertapenem (32%), while a higher percentage of water isolates displayed resistance to tetracycline (55%) and ciprofloxacin (71%). Half of all isolates displayed extended spectrum beta-lactamase (ESBL) production phenotypically (n = 105/211; 50%), with blaCTX-M detected in 99/105 isolates by PCR. Carbapenemase genes were identified in 11 isolates (6 sewage, 5 water). The most common variant was blaOXA-48 (n=6), followed by blaNDM-5 (n=2) and blaKPC-2 (n=2). Whole genome sequencing analysis revealed numerous different sequence types in circulation in both waters and sewage including E. coli ST131 (n=15), ST38 (n=8), ST10 (n=4) along with Klebsiella ST405 (n=3) and ST11 (n=2). Core genome MLST (cgMLST) comparisons uncovered three highly similar Klebsiella isolates originating from hospital sewage and two nearby waters. The Klebsiella isolates from an estuary and seawater displayed 99.1% and 98.8% cgMLST identity to the hospital sewage isolate respectively. In addition, three pairs of E. coli isolates from different waters also revealed cgMLST similarities, indicating widespread dissemination and persistence of certain strains in the aquatic environment. These findings highlight the need for routine monitoring of water bodies used for recreational and drinking purposes for the presence of multi-drug resistant organisms.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

Genomic surveillance of Escherichia coli ST131 identifies local expansion and serial replacement of subclones.

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum ß-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short- and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that Clade C was the most prevalent (686 out of 794 isolates, 86 %) of the three major ST131 clades circulating worldwide (A with fimH41, B with fimH22, C with fimH30), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in c. 1985 and subsequently acquired different ESBL gene variants (blaCTX-M-14 vs blaCTX-M-15). An ISEcp1-mediated transposition of the blaCTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of blaCTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10), highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments.

Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: an inter-laboratory study.

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a 'one-stop' test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams ('participants') were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.