Antimicrobial resistance threatens to take 10 million lives per year by 2050. It is a recognised global health crisis and understanding the historic and current spread of resistance determinants is important for informing surveillance and control measures. The 'inheritance' of resistance is difficult to track because horizontal transfer is common. Antimicrobial resistance genes (ARGs) spread rapidly between bacteria, plasmids and chromosomes due to different mobile genetic elements (MGEs). This movement can increase the range of species carrying an ARG, simplify acquisition of multi-resistance, or otherwise alter the selective advantage associated with carriage of the ARG. MGE activity is therefore a significant factor in understanding routes of ARG dissemination. Characterising the combinations of MGEs contributing to the movement of individual ARGs is crucial. Each MGE category has unique genetic characteristics, and distinct impacts on the location and expression of associated ARGs. Here, the ways in which MGEs can meaningfully associate with ARGs are discussed. Approaches for extracting information about MGE associations from bacterial genome sequences are also considered. Accurate and informative annotations of the genetic contexts of relevant ARGs provide crucial insight into the presence of MGEs and their locations relative to ARGs. Combining this genomic information with knowledge about relevant biological processes allows more accurate conclusions to be drawn about transmission and dissemination of ARGs.

Streptococcus pneumoniae causes otitis media and severe diseases including pneumonia, meningitis and bacteraemia. The rise of antimicrobial resistance (AMR) in S. pneumoniae, facilitated by mobile genetic elements (MGEs), complicates infection treatment. While pneumococcal conjugate vaccine (PCV) deployment has reduced disease burden, non-vaccine serotypes (NVTs) have increased and now cause invasive disease. Although PCV reduced the overall AMR incidence, AMR prevalence among NVT pneumococci has increased, creating dual challenges of MGE-driven AMR spread and serotype replacement. In this review, we analysed geographical patterns of serotype replacement and the role of MGE-driven AMR in S. pneumoniae using predefined search terms related to pneumococcus, MGEs and serotype replacement. Search outputs were managed through COVIDENCE. We de-duplicated 3,634 articles, screened 2,085 by title/abstract, assessed 423 based on exclusion criteria, reviewed 298 full texts and included 70 studies meeting our inclusion criteria. Global data revealed reductions in vaccine serotypes following vaccination, with concurrent NVT increases. Tn916-like and Tn5253-like integrative and conjugative elements (ICEs) were associated with tetracycline and macrolide resistance mobilization. Multidrug-resistant NVTs (15A, 15C, 23A, 34 and 35B) continue emerging globally. Our analysis further reinforces other findings that while PCV implementation has successfully reduced vaccine serotype pneumococcal prevalence globally, this success is accompanied by substantial serotype replacement across all continents. This shifting landscape is further complicated by the widespread presence of MGEs mediating AMR in both vaccine and NVTs, particularly through Tn916-like and Tn5253-like ICEs. These dual challenges underscore the urgent need for improved antimicrobial stewardship programmes and the development of serotype-independent vaccines.

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants. They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.

Several bioinformatics genotyping algorithms are now commonly used to characterize antimicrobial resistance (AMR) gene profiles in whole-genome sequencing (WGS) data, with a view to understanding AMR epidemiology and developing resistance prediction workflows using WGS in clinical settings. Accurately evaluating AMR in Enterobacterales, particularly Escherichia coli , is of major importance, because this is a common pathogen. However, robust comparisons of different genotyping approaches on relevant simulated and large real-life WGS datasets are lacking. Here, we used both simulated datasets and a large set of real E. coli WGS data (n=1818 isolates) to systematically investigate genotyping methods in greater detail. Simulated constructs and real sequences were processed using four different bioinformatic programs (ABRicate, ARIBA, KmerResistance and SRST2, run with the ResFinder database) and their outputs compared. For simulation tests where 3079 AMR gene variants were inserted into random sequence constructs, KmerResistance was correct for 3076 (99.9 %) simulations, ABRicate for 3054 (99.2 %), ARIBA for 2783 (90.4 %) and SRST2 for 2108 (68.5 %). For simulation tests where two closely related gene variants were inserted into random sequence constructs, KmerResistance identified the correct alleles in 35 338/46 318 (76.3 %) simulations, ABRicate identified them in 11 842/46 318 (25.6 %) simulations, ARIBA identified them in 1679/46 318 (3.6 %) simulations and SRST2 identified them in 2000/46 318 (4.3 %) simulations. In real data, across all methods, 1392/1818 (76 %) isolates had discrepant allele calls for at least 1 gene. In addition to highlighting areas for improvement in challenging scenarios, (e.g. identification of AMR genes at <10× coverage, identifying multiple closely related AMR genes present in the same sample), our evaluations identified some more systematic errors that could be readily soluble, such as repeated misclassification (i.e. naming) of genes as shorter variants of the same gene present within the reference resistance gene database. Such naming errors accounted for at least 2530/4321 (59 %) of the discrepancies seen in real data. Moreover, many of the remaining discrepancies were likely ‘artefactual’, with reporting of cut-off differences accounting for at least 1430/4321 (33 %) discrepants. Whilst we found that comparing outputs generated by running multiple algorithms on the same dataset could identify and resolve these algorithmic artefacts, the results of our evaluations emphasize the need for developing new and more robust genotyping algorithms to further improve accuracy and performance.

SUMMARY:With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining a long-read-first assembly approach with short-read polishing. However, existing methods for assembling bacterial plasmids from long-read-first assemblies often misassemble or even miss bacterial plasmids entirely and accordingly require manual curation. Plassembler was developed to provide a tool that automatically assembles and outputs bacterial plasmids using a hybrid assembly approach. It achieves increased accuracy and computational efficiency compared to the existing gold standard tool Unicycler by removing chromosomal reads from the input read sets using a mapping approach. AVAILABILITY AND IMPLEMENTATION:Plassembler is implemented in Python and is installable as a bioconda package using 'conda install -c bioconda plassembler'. The source code is available on GitHub at https://github.com/gbouras13/plassembler. The full benchmarking pipeline can be found at https://github.com/gbouras13/plassembler_simulation_benchmarking, while the benchmarking input FASTQ and output files can be found at https://doi.org/10.5281/zenodo.7996690.

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

INTRODUCTION:Risk factors for carbapenemase-producing Enterobacterales (CPE) acquisition/infection and associated clinical outcomes have been evaluated in the context of clonal, species-specific outbreaks. Equivalent analyses for complex, multi-species outbreaks, which are increasingly common, are lacking. METHODS:Between December 2010 and January 2017, a case-control study of Klebsiella pneumoniae carbapenemase (KPC)-producing organism (KPCO) acquisition was undertaken using electronic health records from inpatients in a US academic medical centre and long-term acute care hospital (LTACH) with ongoing multi-species KPCO transmission despite a robust CPE screening programme. Cases had a first KPCO-positive culture >48 h after admission, and included colonizations and infections (defined by clinical records). Controls had at least two negative perirectal screens and no positive cultures. Risk factors for KPCO acquisition, first infection following acquisition, and 14-day mortality following each episode of infection were identified using multi-variable logistic regression. RESULTS:In 303 cases (89 with at least one infection) and 5929 controls, risk factors for KPCO acquisition included: longer inpatient stay, transfusion, complex thoracic pathology, mechanical ventilation, dialysis, and exposure to carbapenems and β-lactam/β-lactamase inhibitors. Exposure to other KPCO-colonized patients was only a risk factor for acquisition in a single unit, suggesting that direct patient-to-patient transmission did not play a major role. There were 15 species of KPCO; 61 (20%) cases were colonized/infected with more than one species. Fourteen-day mortality following non-urinary KPCO infection was 20% (20/97 episodes) and was associated with failure to achieve source control. CONCLUSIONS:Healthcare exposures, antimicrobials and invasive procedures increased the risk of KPCO colonization/infection, suggesting potential targets for infection control interventions in multi-species outbreaks. Evidence for patient-to-patient transmission was limited.

With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI's AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 E. coli bloodstream infection isolates from Oxfordshire, UK, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity 23% (78/339)) but improved when genetic features associated with penicillinase hyper-production (e.g. promoter mutations, copy number estimates) were considered (sensitivity 82% (277/339); p<0.0001). Most discrepancies occurred in isolates with peri-breakpoint MICs. We investigated two potential causes; the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

ABSTRACTExtended-spectrum cephalosporin resistance (ESC-R) in Escherichia coli and Klebsiella pneumoniae is a healthcare threat; high gastrointestinal carriage rates are reported from South-east Asia. Colonisation prevalence data in Cambodia are lacking. We determined gastrointestinal colonisation prevalence of ESC-resistant E. coli (ESC-R-EC) and K. pneumoniae (ESC-R-KP) in Cambodian children/adolescents and associated risk factors; characterised relevant resistance genes, their genetic contexts, and the genetic relatedness of ESC-R strains using whole genome sequencing (WGS). Faeces and questionnaire data were obtained from individuals <16 years in northwestern Cambodia, 2012. WGS of cultured ESC-R-EC/KP was performed (Illumina). Maximum likelihood phylogenies were used to characterise relatedness of isolates; ESC-R-associated resistance genes and their genetic contexts were identified from de novo assemblies using BLASTn and automated/manual annotation. 82/148 (55%) of children/adolescents were ESC-R-EC/KP colonised; 12/148 (8%) were co-colonised with both species. Independent risk factors for colonisation were hospitalisation (OR: 3.12, 95%, CI [1.52-6.38]) and intestinal parasites (OR: 3.11 [1.29-7.51]); school attendance conferred decreased risk (OR: 0.44 [0.21-0.92]. ESC-R strains were diverse; the commonest ESC-R mechanisms were blaCTX-M 1 and 9 sub-family variants. Structures flanking these genes were highly variable, and for blaCTX-M-15,-55and-27, frequently involved IS26. Chromosomal blaCTX-M integration was common in E. coli. Gastrointestinal ESC-R-EC/KP colonisation is widespread in Cambodian children/adolescents; hospital admission and intestinal parasites are independent risk factors. The genetic contexts of blaCTX-M are highly mosaic, consistent with rapid horizontal exchange. Chromosomal integration of blaCTX-M may result in stable propagation in these community-associated pathogens.

Abstract Background Carbapenemase-producing Enterobacteriaceae can form a reservoir in hospital wastewater biofilms. Klebsiella quasipneumoniae is increasingly recognized as an emerging nosocomial threat, frequently carrying antimicrobial resistance (AMR) genes on plasmids. The dynamics of AMR gene and plasmid gain/loss over time in this species remain unclear. Methods Klebsiella pneumoniae carbapenemase producing-K. quasipneumoniae (KPC-Kq) isolates from patients and wastewater sites from drains and toilets were sequenced (Illumina). Sequence assemblies (SPAdes) were probed in silico for AMR genes and plasmid Incompatibility types (using AMRFinder and PlasmidFinder databases, respectively). For related isolates (<100 SNV) cultured from the same sites longitudinally, we compared the accumulation of AMR genes in patients and environmental reservoirs over time. Results From 2009 to 2016 there were a total of 15 KPC-Kq isolates from 8 patients and 17 environmental isolates from 11 rooms. The mean number of resistance genes identified in patients and environmental isolates were 15 and 14, respectively (P = NS), with five resistance genes carried by all isolates including blaKPC. There was an average of 4.4 unique incompatibility types from patients and 4.0 from the environment (P = NS). For the longitudinal subset, there were 17 related isolates from two patients and two sink drains. One hospitalized patient with repeated antimicrobial exposure had a KPC-Kq initial isolate with 3 plasmid types and 13 AMR genes and died one year later with a KPC-Kq isolated from blood with 11 plasmid types and 25 AMR genes. The other patient was primarily an outpatient with little antimicrobial exposure. His KPC-Kq lost 1 plasmid and 3 AMR genes over 15 months. One KPC-Kq strain in the environment lost 3 plasmid types and 8 AMR genes over 4 months; the other was unchanged over 5 months. Conclusion KPC-Kq has been seen in both patients and the environment for several years at our institution. Sequencing of longitudinal isolates revealed that under antimicrobial pressure a patient KPC-Kq accumulated multiple plasmids and AMR genes. This same accumulation was not witnessed environmental sites over time although the numbers are small and will require confirmatory work. Disclosures All authors: No reported disclosures.

AbstractWith multidrug resistant (MDR) Enterobacteriales on the rise, a non-toxic agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-E. coliin a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with multiple strains and species of KPC-producing clinical isolates collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole genome sequencing (WGS) data using NCBI’s AMRFinder and custom HMM search. Susceptibility testing was performed using glucose-6-phosphate supplemented fosfomycin E-Test and Kirby-Bauer disk diffusion (DD) assays, and compared to agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints forE. coliwere applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by E-Test, 70% (67/96) by DD, and 88% (84/96) by agar dilution.FosAwas detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, andfosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibitfosAand showed significant increases in the zone diameter of DD testing for isolates that werefosA-positive compared tofosA-negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate thatfosAinfluences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory when compared to agar dilution. Further research is needed to determine the impact offosAon clinical outcomes.

As the spread of antimicrobial resistance (AMR) genes becomes an increasing global threat, improved understanding of mobile genetic elements which contribute to the spread of antimicrobial resistance genes, becomes more critical. We created transconjugants from the mating of three chromosomally isogenic Klebsiella pneumoniae carbapenemase (blaKPC) positive Citrobacter freundii isolates with a laboratory strain of Escherichia coli and evaluated the movement of small cryptic plasmids (SCPs), p3223 and p1916, when larger blaKPC-plasmids were transferred. In all of the 143 transconjugants, multiple plasmids, both large and small, transferred with each mating. When two blaKPC-plasmids were present in the host, frequently (87%; 98/113) both would be transferred during mating. p3223 is found in a wide range of bacterial hosts that harbor AMR genes; p1916 has been identified in only a limited number of publicly available sequences to date. From our evaluation, there is still much to learn about SCPs, and the high rate of co-transfer of multiple plasmids from real-world carbapenemase-producing Enterobacteriales.

BACKGROUND Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden. OBJECTIVES To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon. METHODS Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing. RESULTS CPE populations increased during inoculation, plateauing at ∼105 log10 cfu/mL in both models and persisting throughout the experiments (>65 days), with no evidence of CPE 'washout'. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742 bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (∼170 kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model). CONCLUSIONS CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials.

The incidence of Klebsiella pneumoniae infections that are resistant to carbapenems, a last-line class of antibiotics, has been rapidly increasing. The primary mechanism of carbapenem resistance is production of carbapenemase enzymes, which are most frequently encoded on plasmids by bla OXA-48-like, bla VIM, bla NDM and bla KPC genes. Using short-read sequence data, we previously analysed genomes of 1717 isolates from the K. pneumoniae species complex submitted during the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE). Here, we investigated the diversity, prevalence and transmission dynamics of carbapenemase-encoding plasmids using long-read sequencing of representative isolates ( n =79) from this collection in combination with short-read data from all isolates. We highlight three major patterns by which carbapenemase genes have disseminated via plasmids. First, bla OXA-48-like genes have spread across diverse lineages primarily via a highly conserved, epidemic pOXA-48-like plasmid. Second, bla VIM and bla NDM genes have spread via transient associations of diverse plasmids with numerous lineages. Third, bla KPC genes have transmitted predominantly by stable association with one clonal lineage (ST258/512) despite frequent mobilisation between pre-existing yet diverse plasmids within the lineage. Despite contrasts in these three modes of carbapenemase gene spread, which can be summarised as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage, all are underpinned by significant propagation along high-risk clonal lineages.

Much of the worldwide dissemination of antibiotic resistance has been driven by resistance gene associations with mobile genetic elements (MGEs), such as plasmids and transposons. Although increasing, our understanding of resistance spread remains relatively limited, as methods for tracking mobile resistance genes through multiple species, strains and plasmids are lacking. We have developed a bioinformatic pipeline for tracking variation within, and mobility of, specific transposable elements (TEs), such as transposons carrying antibiotic-resistance genes. TETyper takes short-read whole-genome sequencing data as input and identifies single-nucleotide mutations and deletions within the TE of interest, to enable tracking of specific sequence variants, as well as the surrounding genetic context(s), to enable identification of transposition events. A major advantage of TETyper over previous methods is that it does not require a genome reference. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of over 3000 publicly available Illumina datasets containing bla KPC. This revealed surprising diversity, with over 200 distinct flanking genetic contexts for Tn4401, indicating high levels of transposition. Integration of sample metadata revealed insights into associations between geographic locations, host species, Tn4401 sequence variants and flanking genetic contexts. To demonstrate the ability of TETyper to cope with high-copy-number TEs and to track specific short-term evolutionary changes, we also applied it to the insertion sequence IS26 within a defined K. pneumoniae outbreak. TETyper is implemented in python and is freely available at https://github.com/aesheppard/TETyper.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intra-species transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, bla KPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10 and five-year periods respectively. There were 32 bla KPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from eight patients and 11 sink drains, with evidence for seven separate bla KPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies quasipneumoniae (n=23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then probable transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique bla KPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

BACKGROUND:Candida auris is an emerging and multidrug-resistant pathogen. Here we report the epidemiology of a hospital outbreak of C. auris colonization and infection. METHODS:After identification of a cluster of C. auris infections in the neurosciences intensive care unit (ICU) of the Oxford University Hospitals, United Kingdom, we instituted an intensive patient and environmental screening program and package of interventions. Multivariable logistic regression was used to identify predictors of C. auris colonization and infection. Isolates from patients and from the environment were analyzed by whole-genome sequencing. RESULTS:A total of 70 patients were identified as being colonized or infected with C. auris between February 2, 2015, and August 31, 2017; of these patients, 66 (94%) had been admitted to the neurosciences ICU before diagnosis. Invasive C. auris infections developed in 7 patients. When length of stay in the neurosciences ICU and patient vital signs and laboratory results were controlled for, the predictors of C. auris colonization or infection included the use of reusable skin-surface axillary temperature probes (multivariable odds ratio, 6.80; 95% confidence interval [CI], 2.96 to 15.63; P<0.001) and systemic fluconazole exposure (multivariable odds ratio, 10.34; 95% CI, 1.64 to 65.18; P=0.01). C. auris was rarely detected in the general environment. However, it was detected in isolates from reusable equipment, including multiple axillary skin-surface temperature probes. Despite a bundle of infection-control interventions, the incidence of new cases was reduced only after removal of the temperature probes. All outbreak sequences formed a single genetic cluster within the C. auris South African clade. The sequenced isolates from reusable equipment were genetically related to isolates from the patients. CONCLUSIONS:The transmission of C. auris in this hospital outbreak was found to be linked to reusable axillary temperature probes, indicating that this emerging pathogen can persist in the environment and be transmitted in health care settings. (Funded by the National Institute for Health Research Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University and others.).

Infections caused by Streptococcus agalactiae, the Group B streptococcus (GBS), are not associated with food; however, a fish-borne outbreak of GBS ST283 infections was reported in Singapore in 2015, including community-associated meningitis in adults with few co-morbidities [[1]Kalimuddin S. Chen S.L. Lim C.T.K. Koh T.H. Tan T.Y. Kam M. et al.2015 epidemic of severe Streptococcus agalactiae sequence type 283 infections in Singapore associated with the consumption of raw freshwater fish: a detailed analysis of clinical, epidemiological, and bacterial sequencing data.Clin Infect Dis. 2017; 64: S145-S152Crossref PubMed Scopus (57) Google Scholar]. These were similar to meningitis cases seen in Singapore in 1998 [[2]Wilder-Smith E. Chow K.M. Kay R. Ip M. Tee N. Group B streptococcal meningitis in adults: recent increase in Southeast Asia.Aust N Z J Med. 2000; 30: 462-465Crossref PubMed Scopus (25) Google Scholar], although they had been reported as ST11 after standard Sanger sequencing of seven loci [[3]Jones N. Bohnsack J.F. Takahashi S. Oliver K.A. Chan M.S. Kunst F. et al.Multilocus sequence typing system for Group B Streptococcus.J Clin Microbiol. 2003; 41: 2530-2536Crossref PubMed Scopus (455) Google Scholar]. Only two reports of ST283 appeared in the human literature before 2015: one from Hong Kong reporting invasive disease in adults and one from France of two osteoarticular infections [[4]Salloum M. van der Mee-Marquet N. Domelier A.S. Arnault L. Quentin R. Molecular characterization and prophage DNA contents of Streptococcus agalactiae strains isolated from adult skin and osteoarticular infections.J Clin Microbiol. 2010; 48: 1261-1269Crossref PubMed Scopus (17) Google Scholar]. Neither reported further epidemiology. ST11 is a single locus variant of ST283 (it only differs in one of seven housekeeping genes used to determine sequence type). We compared ST11 and ST283 strains by whole genome sequencing to achieve higher resolution than multilocus sequence typing (MLST). Whole genome sequencing raw reads for five of the 1998 GBS ST11 isolates were collected (GenBank accessions SRR6282417–SRR6282421 (under BioProject PRJNA417692)), and compared with existing sequences. All data sets labelled as Streptococcus agalactiae found in GenBank Microbial Genomes and the Short Read Archive as of 25 April 2016 were also downloaded. The MLST types were assigned using a custom blast-based script for assembled genomes and using SRST2 for short read data. MLST profiles were downloaded from https://pubmlst.org/sagalactiae/ on 19 April 2016. Single nucleotide polymorphism (SNP) positions for assembled genomes were inferred by using nucmer and show-snps from the MUMmer package (version 3.23), using the SG-M1 genome as a reference. The SNP positions for short read data sets were called by mapping reads to the SG-M1 genome with bwa (version 0.7.10), then calling SNPs with LoFreq (version 2.1.2) using recommended default parameters (local realignment, minimum coverage 10, filter against strand bias with false discovery rate method). SNP positions for all data sets were integrated into a single VCF file. This was processed with the SNPRelate package into GDS format, from which only positions with biallelic SNPs were extracted. SNPRelate was further used to calculate a pairwise dissimilarity matrix. An unrooted neighbor-joining tree was generated from this dissimilarity matrix using the ape package and visualized using the ggtree package (DOI: 10.1111/2041-210X.12628) in R (version 3.4.2) (see Supplementary material, Appendix S1, for methodological references). The five GBS 1998 isolates originally reported as ST11 were in fact ST283 based both on direct typing from short reads and on allele sequences extracted from de novo assemblies. The only difference is that ST11 has pheS_3, whereas ST283 has pheS_5; these pheS alleles differ by four nucleotide differences in the middle third of the gene. We hypothesize that ST11 was misdefined due to a clerical error that inserted pheS_3 in place of pheS_5, so ST11 was erroneously defined as 9-3-7-1-3-3-2 when it should have been 9-5-7-1-3-3-2, which was subsequently assigned ST283. The original sequence data from which MLST alleles were called for the five ‘ST11’ isolates were unavailable. We therefore examined publicly available databases for further evidence. Among all 1088 GBS MLST profiles, ST11 is the only sequence type that has a combination of pheS_3 with adhP_9. In contrast, ST283 is one of five MLSTs that have both pheS_5 and adhP_9. If ST11 were excluded, there would be no overlap in adhP alleles between pheS_3 and pheS_5 MLSTs. In summary, pheS alleles 3 and 5 have totally different associations with adhP alleles. We performed MLST typing on 1411 publicly available GBS whole genome sequencing short read data sets. None of these were typed as ST11. Only 26 strains had pheS_3, and 188 strains had pheS_5 (including all ST283 isolates). All isolates carrying pheS_3 formed a closely related and apparently monophyletic clade on a reference-based phylogenetic tree, while the pheS_5-carrying isolates formed a separate, apparently unrelated monophyletic clade (Fig. 1a). Notably, we typed one fish isolate as ST491, which is a single locus variant of ST283 that differs by a single nucleotide in the tkt gene; it falls clearly within the ST283 clade (blue dot in Fig. 1b). Analysis of pairwise SNP differences show that only 10–28 SNPs distinguish the five original ST11 isolates from 1998 from each other while 35–47 SNPs distinguish the 1998 isolates from those isolated in 2015. The 1998 isolates fall within the ST283 clade (red dots in Fig. 1b, red branches in Fig. 1c). We conclude that the allele combination defined by ST11 may not exist and that an occult source of ST283 may have persisted since 1998, if not before. The implication is that there may have been unrecognized food-borne invasive GBS ST283 disease in Singapore for over two decades. Considering the freshwater fish implicated in the 2015 Singapore outbreak were imported from surrounding countries, that Hong Kong reported a similar experience with ST283 between 1995 and 2012 [[5]Ip M. Ang I. Fung K. Liyanapathirana V. Luo M.J. Lai R. Hypervirulent clone of Group B streptococcus serotype III sequence type 283, Hong Kong, 1993–2012.Emerg Infect Dis. 2016; 22: 1800-1803Crossref PubMed Scopus (21) Google Scholar] and that ST283 and the single locus variant ST491 were reported from one fish from Thailand and Vietnam, respectively [[6]Delannoy C.M. Crumlish M. Fontaine M.C. Pollock J. Foster G. Dagleish M.P. et al.Human Streptococcus agalactiae strains in aquatic mammals and fish.BMC Microbiol. 2013; 13: 41Crossref PubMed Scopus (148) Google Scholar], we suspect that ST283 may be present throughout the South-East Asia region, in both humans and fish, escaping detection due to the unrecognized epidemiology and lack of regional GBS typing data. TB/SC have a pending patent: A method of detecting Group B Streptococcus bacteria, related nucleotides and kits. No external funding was received. The correction of ST11 to ST283 was briefly presented at the International Symposium on Streptococcus agalactiae Disease, Cape Town, on 23 February 2018. TB created the hypothesis and organized the collaboration; SC performed the bioinformatics analysis; AS and NJ sequenced the isolates. All authors contributed to the manuscript. The following is the supplementary data related to this article: Download .docx (.02 MB) Help with docx files Appendix S1Methodological details with references.