Comparison of contemporary invasive and non-invasive Streptococcus pneumoniae isolates reveals new insights into circulating anti-microbial resistance determinants.

Streptococcus pneumoniae is a major human pathogen with a high burden of disease. Non-invasive isolates (those found in non-sterile sites) are thought to be a key source of invasive isolates (those found in sterile sites) and a reservoir of anti-microbial resistance (AMR) determinants. Despite this, pneumococcal surveillance has almost exclusively focused on invasive isolates. We aimed to compare contemporaneous invasive and non-invasive isolate populations to understand how they interact and identify differences in AMR gene distribution. We used a combination of whole-genome sequencing and phenotypic anti-microbial susceptibility testing and a data set of invasive (n = 1,288) and non-invasive (n = 186) pneumococcal isolates, collected in Victoria, Australia, between 2018 and 2022. The non-invasive population had increased levels of antibiotic resistance to multiple classes of antibiotics including beta-lactam antibiotics penicillin and ceftriaxone. We identified genomic intersections between the invasive and non-invasive populations and no distinct phylogenetic clustering of the two populations. However, this analysis revealed sub-populations overrepresented in each population. The sub-populations that had high levels of AMR were overrepresented in the non-invasive population. We determined that WamR-Pneumo was the most accurate in silico tool for predicting resistance to the antibiotics tested. This tool was then used to assess the allelic diversity of the penicillin-binding protein genes, which acquire mutations leading to beta-lactam antibiotic resistance, and found that they were highly conserved (≥80% shared) between the two populations. These findings show the potential of non-invasive isolates to serve as reservoirs of AMR determinants.

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae.

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K. pneumoniae lineages or 'clones' that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated ('hypervirulent') clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K. pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K. pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Search and Contain: Impact of an Integrated Genomic and Epidemiological Surveillance and Response Program for Control of Carbapenemase-producing Enterobacterales.

BACKGROUND: Multiresistant organisms (MROs) pose a critical threat to public health. Population-based programs for control of MROs such as carbapenemase-producing Enterobacterales (CPE) have emerged and evaluation is needed. We assessed the feasibility and impact of a statewide CPE surveillance and response program deployed across Victoria, Australia (population 6.5 million). METHODS: A prospective multimodal intervention including active screening, carrier isolation, centralized case investigation, and comparative pathogen genomics was implemented. We analyzed trends in CPE incidence and clinical presentation, risk factors, and local transmission over the program's first 3 years (2016-2018). RESULTS: CPE case ascertainment increased over the study period to 1.42 cases/100 000 population, linked to increased screening without a concomitant rise in active clinical infections (0.45-0.60 infections/100 000 population, P = .640). KPC-2 infection decreased from 0.29 infections/100 000 population prior to intervention to 0.03 infections/100 000 population in 2018 (P = .003). Comprehensive case investigation identified instances of overseas community acquisition. Median time between isolate referral and genomic and epidemiological assessment for local transmission was 11 days (IQR, 9-14). Prospective surveillance identified numerous small transmission networks (median, 2; range, 1-19 cases), predominantly IMP and KPC, with median pairwise distance of 8 (IQR, 4-13) single nucleotide polymorphisms; low diversity between clusters of the same sequence type suggested genomic cluster definitions alone are insufficient for targeted response. CONCLUSIONS: We demonstrate the value of centralized CPE control programs to increase case ascertainment, resolve risk factors, and identify local transmission through prospective genomic and epidemiological surveillance; methodologies are transferable to low-prevalence settings and MROs globally.

Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients.

BACKGROUND: Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. METHODS: We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. RESULTS: Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). CONCLUSIONS: 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

Genomics for Molecular Epidemiology and Detecting Transmission of Carbapenemase-Producing Enterobacterales in Victoria, Australia, 2012 to 2016.

Carbapenemase-producing Enterobacterales (CPE) are being increasingly reported in Australia, and integrated clinical and genomic surveillance is critical to effectively manage this threat. We sought to systematically characterize CPE in Victoria, Australia, from 2012 to 2016. Suspected CPE were referred to the state public health laboratory in Victoria, Australia, from 2012 to 2016 and examined using phenotypic, multiplex PCR and whole-genome sequencing (WGS) methods and compared with epidemiological metadata. Carbapenemase genes were detected in 361 isolates from 291 patients (30.8% of suspected CPE isolates), mostly from urine (42.1%) or screening samples (34.8%). IMP-4 (28.0% of patients), KPC-2 (25.3%), NDM (24.1%), and OXA carbapenemases (22.0%) were most common. Klebsiella pneumoniae (48.8% of patients) and Escherichia coli (26.1%) were the dominant species. Carbapenemase-inactivation method (CIM) testing reliably detected carbapenemase-positive isolates (100% sensitivity, 96.9% specificity), identifying an additional five CPE among 159 PCR-negative isolates (IMI and SME carbapenemases). When epidemiologic investigations were performed, all pairs of patients designated "highly likely" or "possible" local transmission had ≤23 pairwise single-nucleotide polymorphisms (SNPs) by genomic transmission analysis; conversely, all patient pairs designated "highly unlikely" local transmission had ≥26 pairwise SNPs. Using this proposed threshold, possible local transmission was identified involving a further 16 patients for whom epidemiologic data were unavailable. Systematic application of genomics has uncovered the emergence of polyclonal CPE as a significant threat in Australia, providing important insights to inform local public health guidelines and interventions. Using our workflow, pairwise SNP distances between CPE isolates of ≤23 SNPs suggest local transmission.

Antimicrobial-Resistant Klebsiella pneumoniae Carriage and Infection in Specialized Geriatric Care Wards Linked to Acquisition in the Referring Hospital.

Background: Klebsiella pneumoniae is a leading cause of extended-spectrum ß-lactamase (ESBL)-producing hospital-associated infections, for which elderly patients are at increased risk. Methods: We conducted a 1-year prospective cohort study, in which a third of patients admitted to 2 geriatric wards in a specialized hospital were recruited and screened for carriage of K. pneumoniae by microbiological culture. Clinical isolates were monitored via the hospital laboratory. Colonizing and clinical isolates were subjected to whole-genome sequencing and antimicrobial susceptibility testing. Results: K. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8%, and ESBL carriage 1.7%, and the incidence of K. pneumoniae infection was 1.2%. The isolates were diverse, and most patients were colonized or infected with a unique phylogenetic lineage, with no evidence of transmission in the wards. ESBL strains carried blaCTX-M-15 and belonged to clones associated with hospital-acquired ESBL infections in other countries (sequence type [ST] 29, ST323, and ST340). One also carried the carbapenemase blaIMP-26. Genomic and epidemiological data provided evidence that ESBL strains were acquired in the referring hospital. Nanopore sequencing also identified strain-to-strain transmission of a blaCTX-M-15 FIBK/FIIK plasmid in the referring hospital. Conclusions: The data suggest the major source of K. pneumoniae was the patient's own gut microbiome, but ESBL strains were acquired in the referring hospital. This highlights the importance of the wider hospital network to understanding K. pneumoniae risk and infection prevention. Rectal screening for ESBL organisms on admission to geriatric wards could help inform patient management and infection control in such facilities.

Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads.

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate "hybrid" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health.

Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

Gastrointestinal Carriage Is a Major Reservoir of Klebsiella pneumoniae Infection in Intensive Care Patients.

BACKGROUND: Klebsiella pneumoniae is an opportunistic pathogen and leading cause of hospital-associated infections. Intensive care unit (ICU) patients are particularly at risk. Klebsiella pneumoniae is part of the healthy human microbiome, providing a potential reservoir for infection. However, the frequency of gut colonization and its contribution to infections are not well characterized. METHODS: We conducted a 1-year prospective cohort study in which 498 ICU patients were screened for rectal and throat carriage of K. pneumoniae shortly after admission. Klebsiella pneumoniae isolated from screening swabs and clinical diagnostic samples were characterized using whole genome sequencing and combined with epidemiological data to identify likely transmission events. RESULTS: Klebsiella pneumoniae carriage frequencies were estimated at 6% (95% confidence interval [CI], 3%-8%) among ICU patients admitted direct from the community, and 19% (95% CI, 14%-51%) among those with recent healthcare contact. Gut colonization on admission was significantly associated with subsequent infection (infection risk 16% vs 3%, odds ratio [OR] = 6.9, P < .001), and genome data indicated matching carriage and infection isolates in 80% of isolate pairs. Five likely transmission chains were identified, responsible for 12% of K. pneumoniae infections in ICU. In sum, 49% of K. pneumoniae infections were caused by the patients' own unique strain, and 48% of screened patients with infections were positive for prior colonization. CONCLUSIONS: These data confirm K. pneumoniae colonization is a significant risk factor for infection in ICU, and indicate ~50% of K. pneumoniae infections result from patients' own microbiota. Screening for colonization on admission could limit risk of infection in the colonized patient and others.

Carbapenemase-producing enterobacterales colonisation status does not lead to more frequent admissions: a linked patient study.

BACKGROUND: Hospitals in any given region can be considered as part of a network, where facilities are connected to one another - and hospital pathogens potentially spread - through the movement of patients between them. We sought to describe the hospital admission patterns of patients known to be colonised with carbapenemase-producing Enterobacterales (CPE), and compare them with CPE-negative patient cohorts, matched on comorbidity information. METHODS: We performed a linkage study in Victoria, Australia, including datasets with notifiable diseases (CPE notifications) and hospital admissions (admission dates and diagnostic codes) for the period 2011 to 2020. Where the CPE notification date occurred during a hospital admission for the same patient, we identified this as the 'index admission'. We determined the number of distinct health services each patient was admitted to, and time to first admission to a different health service. We compared CPE-positive patients with four cohorts of CPE-negative patients, sampled based on different matching criteria. RESULTS: Of 528 unique patients who had CPE detected during a hospital admission, 222 (42%) were subsequently admitted to a different health service during the study period. Among these patients, CPE diagnosis tended to occur during admission to a metropolitan public hospital (86%, 190/222), whereas there was a greater number of metropolitan private (23%, 52/222) and rural public (18%, 39/222) hospitals for the subsequent admission. Median time to next admission was 4 days (IQR, 0-75 days). Admission patterns for CPE-positive patients was similar to the cohort of CPE-negative patients matched on index admission, time period, and age-adjusted Charlson comorbidity index. CONCLUSIONS: Movement of CPE-positive patients between health services is not a rare event. While the most common movement is from one public metropolitan health service to another, there is also a trend for movement from metropolitan public hospitals into private and rural hospitals. After accounting for clinical comorbidities, CPE colonisation status does not appear to impact on hospital admission frequency or timing. These findings support the potential utility of a centralised notification and outbreak management system for CPE positive patients.

Hidden Resistances: How Routine Whole-Genome Sequencing Uncovered an Otherwise Undetected blaNDM-1 Gene in Vibrio alginolyticus from Imported Seafood.

Vibrio alginolyticus causes vibriosis of marine vertebrates, invertebrates, and humans, and while there have been several reports of multidrug resistance in V. alginolyticus, carbapenem resistance is rare. V. alginolyticus strain AUSMDU00064140 was isolated in Melbourne, Australia, from imported prawns. Routine genomic surveillance detected the presence of a full-length blaNDM-1 gene, subsequently shown to be collocated with additional acquired antimicrobial resistance genes on a resistance cassette on the largest chromosome, flanked by mobilization gene annotations. Comparisons to a previously described V. alginolyticus plasmid, pC1349, revealed differing gene content and arrangements between the resistance cassettes. Phylogenetic analysis was performed against a local and global data set (n = 109), demonstrating that AUSMDU00064140 was distinct and did not cluster with any other strains. Despite the presence of the complete blaNDM-1 gene and positive phenotypic assays for carbapenemase production, carbapenem MICs were low (meropenem MIC ≤0.5 mg/liter). However, it is still possible that this gene may be transferred to another species in the environment or a host, causing phenotypic carbapenem resistance and presenting a risk of great public health concern. IMPORTANCE Carbapenems are last-line antimicrobials, vital for use in human medicine. Antimicrobial resistance determinants such as blaNDM (New Delhi metallo-ß-lactamase producing) genes conferring resistance to the carbapenem class of antimicrobials, are typically found in Enterobacterales (first described in 2009 from a Klebsiella pneumoniae isolate). Our study shows that Vibrio alginolyticus isolated from cooked prawn is able to harbor antimicrobial resistance (AMR) genes of public health concern, specifically a chromosomally located blaNDM-1 gene, and there is the potential for transmission of resistance genes. This may be linked with antimicrobial use in low- and middle-income settings, which has typically been high, unregulated, or not reported. Many countries, including Thailand, have implemented national strategic plans to incorporate the World Health Organization (WHO)'s Global Action Plan (2015) recommendations of a global One Health approach, including increased resources for surveillance of antimicrobial usage and AMR; however, efficient antimicrobial surveillance systems incorporating genomic and phenotypic testing of isolates are still lacking in many jurisdictions.

An ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance.

Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR, an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI's AMRFinderPlus, as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.

Population structure, serotype distribution and antibiotic resistance of Streptococcus pneumoniae causing invasive disease in Victoria, Australia.

Streptococcus pneumoniae is a major human pathogen and can cause a range of conditions from asymptomatic colonization to invasive pneumococcal disease (IPD). The epidemiology and distribution of IPD-causing serotypes in Australia has undergone large changes following the introduction of the 7-valent pneumococcal conjugate vaccine (PCV) in 2005 and the 13-valent PCV in 2011. In this study, to provide a contemporary understanding of the IPD causing population in Victoria, Australia, we aimed to examine the population structure and prevalence of antimicrobial resistance using whole-genome sequencing and comprehensive antimicrobial susceptibility data of 1288 isolates collected between 2018 and 2022. We observed high diversity among the isolates with 52 serotypes, 203 sequence types (STs) and 70 Global Pneumococcal Sequencing Project Clusters (GPSCs) identified. Serotypes contained in the 13v-PCV represented 35.3 % (n=405) of isolates. Antimicrobial resistance (AMR) to at least one antibiotic was identified in 23.8 % (n=358) of isolates with penicillin resistance the most prevalent (20.3 %, n=261 using meningitis breakpoints and 5.1 % n=65 using oral breakpoints). Of the AMR isolates, 28 % (n=101) were multidrug resistant (MDR) (resistant to three or more drug classes). Vaccination status of cases was determined for a subset of isolates with 34 cases classified as vaccine failure events (fully vaccinated IPD cases of vaccine serotype). However, no phylogenetic association with failure events was observed. Within the highly diverse IPD population, we identified six high-risk sub-populations of public health concern characterized by high prevalence, high rates of AMR and MDR, or serotype inclusion in vaccines. High-risk serotypes included serotypes 3, 19F, 19A, 14, 11A, 15A and serofamily 23. In addition, we present our data validating seroBA for in silico serotyping to facilitate ISO-accreditation of this test in routine use in a public health reference laboratory and have made this data set available. This study provides insights into the population dynamics, highlights non-vaccine serotypes of concern that are highly resistant, and provides a genomic framework for the ongoing surveillance of IPD in Australia which can inform next-generation IPD prevention strategies.

Molecular and clinical epidemiology of carbapenem resistant Acinetobacter baumannii ST2 in Oceania: a multicountry cohort study.

Background: Carbapenem resistant Acinetobacter baumannii (CRAb) is categorised by the World Health Organization (WHO) as a pathogen of critical concern. However, little is known about CRAb transmission within the Oceania region. This study addresses this knowledge gap by using molecular epidemiology to characterise the phylogenetic relationships of CRAb isolated in hospitals in Fiji, Samoa, and other countries within the Oceania region including Australia and New Zealand, and India from South Asia. Methods: In this multicountry cohort study, we analysed clinical isolates of CRAb collected from the Colonial War Memorial Hospital (CWMH) in Fiji from January through December 2019 (n = 64) and Tupua Tamasese Mea'ole Hospital (TTMH) in Samoa from November 2017 through June 2021 (n = 32). All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole-genome sequencing. For CWMH, data were collected on clinical and demographic characteristics of patients with CRAb, duration of hospital stay, mortality and assessing the appropriateness of meropenem use from the treated patients who had CRAb infections. To provide a broader geographical context, CRAb strains from Fiji and Samoa were compared with CRAb sequences from Australia collected in 2016-2018 (n = 22), New Zealand in 2018-2021 (n = 13), and India in 2019 (n = 58), a country which has close medical links with Fiji. Phylogenetic relationships of all these CRAb isolates were determined using differences in core genome SNPs. Findings: Of CRAb isolates, 49 (77%) of 64 from Fiji and all 32 (100%) from Samoa belonged to CRAb sequence type 2 (ST2). All ST2 isolates from both countries harboured blaOXA-23, blaOXA-66 and ampC-2 genes, mediating resistance to ß-lactam antimicrobials, including cephalosporins and carbapenems. The blaOXA-23 gene was associated with two copies of ISAba1 insertion element, forming the composite transposon Tn2006, on the chromosome. Two distinct clusters (group 1 and group 2) of CRAb ST2 were detected in Fiji. The first group shared common ancestral linkage to all CRAb ST2 collected from Fiji's historic outbreak in 2016/2017, Samoa, Australia and 54% of total New Zealand isolates; they formed a single cluster with a median (range) SNP difference of 13 (0-102). The second group shared common ancestral linkage to 3% of the total CRAb ST2 isolated from India. Fifty eight of the 64 patients with CRAb infections at the CWMH had their first positive CRAb sample collected 72 h or more following admission. Meropenem use was deemed inappropriate in 15 (48%) of the 31 patients that received treatment with meropenem in Fiji. Other strains of CRAb ST1, ST25, ST107, and ST1112 were also detected in Fiji. Interpretation: We identified unrecognised outbreaks of CRAb ST2 in Fiji and Samoa that linked to strains in other parts of Oceania and South Asia. The existence of Tn2006, containing the blaOXA-23 and ISAba1 insertion element, within CRAb ST2 from Fiji and Samoa indicates the potential for high mobility and dissemination. This raises concerns about unmitigated prolonged outbreaks of CRAb ST2 in the two major hospitals in Fiji and Samoa. Given the magnitude of this problem, there is a need to re-evaluate the current strategies used for infection prevention and control, antimicrobial stewardship, and public health measures locally and internationally. Moreover, a collaborative approach to AMR surveillance within the Oceania region with technical, management and budgetary support systems is required to prevent introduction and control transmission of these highly problematic strains within the island nation health systems. Funding: This project was funded by an Otago Global Health Institute seed grant and Maurice Wilkins Centre of Research Excellence (CoREs) grant (SC0000169653, RO0000002300).

Evaluating the role of asymptomatic throat carriage of Streptococcus pyogenes in impetigo transmission in remote Aboriginal communities in Northern Territory, Australia: a retrospective genomic analysis.

BACKGROUND: Streptococcus pyogenes, or group A Streptococcus (GAS), infections contribute to a high burden of disease in Aboriginal Australians, causing skin infections and immune sequelae such as rheumatic heart disease. Controlling skin infections in these populations has proven difficult, with transmission dynamics being poorly understood. We aimed to identify the relative contributions of impetigo and asymptomatic throat carriage to GAS transmission. METHODS: In this genomic analysis, we retrospectively applied whole genome sequencing to GAS isolates that were collected as part of an impetigo surveillance longitudinal household survey conducted in three remote Aboriginal communities in the Northern Territory of Australia between Aug 6, 2003, and June 22, 2005. We included GAS isolates from all throats and impetigo lesions of people living in two of the previously studied communities. We classified isolates into genomic lineages based on pairwise shared core genomes of more than 99% with five or fewer single nucleotide polymorphisms. We used a household network analysis of epidemiologically and genomically linked lineages to quantify the transmission of GAS within and between households. FINDINGS: We included 320 GAS isolates in our analysis: 203 (63%) from asymptomatic throat swabs and 117 (37%) from impetigo lesions. Among 64 genomic lineages (encompassing 39 emm types) we identified 264 transmission links (involving 93% of isolates), for which the probable source was asymptomatic throat carriage in 166 (63%) and impetigo lesions in 98 (37%). Links originating from impetigo cases were more frequent between households than within households. Households were infected with GAS for a mean of 57 days (SD 39 days), and once cleared, reinfected 62 days (SD 40 days) later. Increased household size and community presence of GAS and scabies were associated with slower clearance of GAS. INTERPRETATION: In communities with high prevalence of endemic GAS-associated skin infection, asymptomatic throat carriage is a GAS reservoir. Public health interventions such as vaccination or community infection control programmes aimed at interrupting transmission of GAS might need to include consideration of asymptomatic throat carriage. FUNDING: Australian National Health and Medical Research Council.

Optimising genomic approaches for identifying vancomycin-resistant Enterococcus faecium transmission in healthcare settings.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation.

Multi-site implementation of whole genome sequencing for hospital infection control: A prospective genomic epidemiological analysis.

Background: Current microbiological methods lack the resolution to accurately identify multidrug-resistant organism (MDRO) transmission, however, whole genome sequencing can identify highly-related patient isolates providing opportunities for precision infection control interventions. We investigated the feasibility and potential impact of a prospective multi-centre genomics workflow for hospital infection control. Methods: We conducted a prospective genomics implementation study across eight Australian hospitals over 15 months (2017,2018), collecting all clinical and screening isolates from inpatients with vanA VRE, MRSA, ESBL Escherichia coli (ESBL-Ec), or ESBL Klebsiella pneumoniae (ESBL-Kp). Genomic and epidemiologic data were integrated to assess MDRO transmission. Findings: In total, 2275 isolates were included from 1970 patients, predominantly ESBL-Ec (40·8%) followed by MRSA (35·6%), vanA VRE (15·2%), and ESBL-Kp (8·3%).Overall, hospital and genomic epidemiology showed 607 patients (30·8%) acquired their MDRO in hospital, including the majority of vanA VRE (266 patients, 86·4%), with lower proportions of ESBL-Ec (186 patients, 23·0%), ESBL-Kp (42 patients, 26·3%), and MRSA (113 patients, 16·3%). Complex patient movements meant the majority of MDRO transmissions would remain undetected without genomic data.The genomics implementation had major impacts, identifying unexpected MDRO transmissions prompting new infection control interventions, and contributing to vanA VRE becoming a notifiable condition. We identified barriers to implementation and recommend strategies for mitigation. Interpretation: Implementation of a multi-centre genomics-informed infection control workflow is feasible and identifies many unrecognised MDRO transmissions. This provides critical opportunities for interventions to improve patient safety in hospitals. Funding: Melbourne Genomics Health Alliance (supported by State Government of Victoria, Australia), and National Health and Medical Research Council (Australia).

Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen.

Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.

Genomic Insights Into Last-Line Antimicrobial Resistance in Multidrug-Resistant Staphylococcus and Vancomycin-Resistant Enterococcus.

Multidrug-resistant Staphylococcus and vancomycin-resistant Enterococcus (VRE) are important human pathogens that are resistant to most clinical antibiotics. Treatment options are limited and often require the use of 'last-line' antimicrobials such as linezolid, daptomycin, and in the case of Staphylococcus, also vancomycin. The emergence of resistance to these last-line antimicrobial agents is therefore of considerable clinical concern. This mini-review provides an overview of resistance to last-line antimicrobial agents in Staphylococcus and VRE, with a particular focus on how genomics has provided critical insights into the emergence of resistant clones, the molecular mechanisms of resistance, and the importance of mobile genetic elements in the global spread of resistance to linezolid.