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Metabolic capacity can vary substantially within a bacterial species, leading to ecological niche separation, as well as differences in virulence and antimicrobial susceptibility. Genome-scale metabolic models are useful tools for studying the metabolic potential of individuals, and with the rapid expansion of genomic sequencing there is a wealth of data that can be leveraged for comparative analysis. However, there exist few tools to construct strain-specific metabolic models at scale. Here we describe Bactabolize (github.com/kelwyres/Bactabolize), a reference-based tool which rapidly produces strain-specific metabolic models and growth phenotype predictions. We describe a pan reference model for the priority antimicrobial-resistant pathogen, Klebsiella pneumoniae (github.com/kelwyres/KpSC-pan-metabolic-model), and a quality control framework for using draft genome assemblies as input for Bactabolize. The Bactabolize-derived model for K. pneumoniae reference strain KPPR1 outperformed the CarveMe-derived model across ≥201 substrate and ≥1220 knockout mutant growth predictions. Novel draft genomes passing our systematically-defined quality control criteria resulted in models with a high degree of completeness (≥99% genes and reactions captured) and high accuracy (mean 0.97, n=10). We anticipate the tools and framework described herein will facilitate large-scale metabolic modelling analyses that broaden our understanding of diversity within bacterial species and inform novel control strategies for priority pathogens.

Background Community-acquired pneumonia is an important cause of morbidity and mortality in adults. Approximately one-third of pneumonia cases can be attributed to the pneumococcus. Pneumococcal conjugate vaccines (PCVs) protect against colonisation with vaccine-type serotypes. The resulting decrease in transmission of vaccine serotypes leads to large indirect effects. There are limited data from developing countries demonstrating the impact of childhood PCV immunisation on adult pneumonia. There are also insufficient data available on the burden and severity of all-cause pneumonia and respiratory syncytial virus (RSV) in adults from low resource countries. There is currently no recommendation for adult pneumococcal vaccination with either pneumococcal polysaccharide vaccine or PCVs in Mongolia. We describe the protocol developed to evaluate the association between childhood 13-valent PCV (PCV13) vaccination and trends in adult pneumonia. Methods PCV13 was introduced into the routine childhood immunisation schedule in Mongolia in a phased manner from 2016. In March 2019 we initiated active hospital-based surveillance for adult pneumonia, with the primary objective of evaluating trends in severe hospitalised clinical pneumonia incidence in adults 18 years and older in four districts of Ulaanbaatar. Secondary objectives include measuring the association between PCV13 introduction and trends in all clinically-defined pneumonia, radiologically-confirmed pneumonia, nasopharyngeal carriage of S. pneumoniae and pneumonia associated with RSV or influenza. Clinical questionnaires, nasopharyngeal swabs, urine samples and chest radiographs were collected from enrolled patients. Retrospective administrative and clinical data were collected for all respiratory disease-related admissions from January 2015 to February 2019. Discussion Establishing a robust adult surveillance system may be an important component of monitoring the indirect impact of PCVs within a country. Monitoring indirect impact of childhood PCV13 vaccination on adult pneumonia provides additional data on the full public health impact of the vaccine, which has implications for vaccine efficiency and cost-effectiveness. Adult surveillance in Mongolia will contribute to the limited evidence available on the burden of pneumococcal pneumonia among adults in low- and middle-income countries, particularly in the Asia-Pacific region. In addition, it is one of the few examples of implementing prospective, population-based pneumonia surveillance to evaluate the indirect impact of PCVs in a resource-limited setting.

Typhoid fever is endemic in some Pacific Island Countries including Fiji and Samoa yet genomic surveillance is not routine in such settings. Previous studies suggested imports of the global H58 clade ofGenomic sequencing approaches were implemented to study the distribution of 255Genomic analyses showedCyclones and flooding drove 'waves' of typhoid outbreaks in Fiji which, through population aggregation, poor sanitation and water safety, and then mobility of the population, spread clones more widely. Minimal international importations of new typhoid clones suggest that targeted local intervention strategies may be useful in controlling endemic typhoid infection. These findings add to our understanding of typhoid transmission networks in an endemic island country with broad implications, particularly across Pacific Island Countries.This work was supported by the Coalition Against Typhoid through the Bill and Melinda Gates Foundation [grant number OPP1017518], the Victorian Government, the National Health and Medical Research Council Australia, the Australian Research Council, and the Fiji Ministry of Health and Medical Services.

Objectives: Nocardia bacteremia is a rare but severe disease associated with high mortality. This systematic review is the largest and most comprehensive review performed over the past 20 years. Methods: A single-center retrospective review of Nocardia bacteremia was performed using hospital microbiology records from January 1, 2010 to December 31, 2017. A systematic literature review was also performed to identify cases of Nocardia bacteremia described in the NCBI PubMed database in English between January 1, 1999 and December 31, 2018. Results: Four new cases of Nocardia bacteremia are described. The systematic review identified 134 cases with sufficient information available for analysis. Of the total 138 cases, the median age was 58 years (interquartile range (IQR) 44–69 years) and 70% were male. Eighty-one percent were immunocompromised (corticosteroid use (49%), hematological malignancy (20%), solid organ transplant (20%), solid organ malignancy (19%), and hematopoietic stem cell transplantation (15%)) and 29% had endovascular devices. Pulmonary infection was the most common concurrent site of clinical disease (67%). The median incubation time to the detection of Nocardia bacteremia was 4 days (IQR 3–6 days). Blood cultures were the only positive microbiological specimen in 38% of cases. The median total duration of treatment was 75 days (IQR 25–182 days). Thirty-day all-cause mortality was 28% and overall all-cause mortality was 40%. Conclusions: Nocardia bacteremia is most frequently identified in immunocompromised patients and those with intravascular devices. Although rare, it represents a serious infection with high associated overall mortality. Keywords: Nocardia, Nocardiosis, Bacteremia, Central line-associated bloodstream infection, Immunocompromise

Clonal complex 398 (CC398) livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been reported worldwide in a variety of food-animal species. Although CC398 is synonymous with LA-MRSA, community-associated MRSA (CA-MRSA) variants have emerged, including the Panton-Valentine leukocidin (PVL)-positive ST398-V and ST398 single-locus variant ST1232-V, and the PVL-negative ST398-V clones. Using comparative genomic analysis, we determined whether ten CC398 MRSA bacteraemia episodes recently identified in Australia were due to LA-MRSA or CA-MRSA CC398. Isolates were sourced from the Australian Group on Antimicrobial Resistance S. aureus surveillance programme and episodes occurred across Australia. Whole-genome sequencing (WGS) and phylogenetic comparison of the ten CC398 bacteraemia isolates with previously published CC398 MRSA whole-genome sequences identified that the Australian CC398 isolates were closely related to the human-associated II-GOI clade and the livestock-associated IIa clade. The identified CC398 MRSA clones were: PVL-positive ST1232-V (5C25), PVL-negative community-associated ST398-V (5C25) and livestock-associated ST398-V (5C25). Our findings demonstrate the importance of using WGS and comparing the sequences with international sequences to distinguish between CC398 CA-MRSA and LA-MRSA and to determine the isolates' origin. Furthermore, our findings suggest that CC398 CA-MRSA has become established in the Australian community and that ST398-V (5C25) LA-MRSA is now widespread in Australian piggeries. Our study emphasises the need for national One Health antimicrobial resistance surveillance programmes to assist in monitoring the ongoing epidemiology of MRSA and other clinically significant antimicrobial-resistant organisms.

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.

Introduction: The confirmation or analysis and exclusion of a diagnosis of neurosyphilis has long presented a challenge for infectious diseases clinicians. The authors reviewed the concordance between cerebrospinal fluid (CSF) analysis and the subsequent antibiotic strategy for patients undergoing evaluation of a diagnosis of neurosyphilis. Methods: All patients with positive serum syphilis serology referred for CSF analysis between January 2009 and May 2016 were included. Indications for CSF analysis were determined by review of the hospital electronic medical records. CSF parameters were determined from the hospital pathology database. Cases were defined as either ‘confirmed’, ‘supportive’ of, or ‘not supportive’ of a diagnosis of neurosyphilis based on existing definitions. Subsequent therapy was defined as for neurosyphilis, late latent primary syphilis or no therapy based on existing guidelines. Results: Of 131 patients reviewed, 95.4% were male and HIV co-infected (74%). A confirmed diagnosis of neurosyphilis was met by fourteen patients (10.7%). All but two of these were treated with a neurosyphilis-directed regimen. Of the 58 patients treated with neurosyphilis antibiotics, 17.2% had no CSF findings suggestive of the diagnosis. Seventy-three patients were not treated for neurosyphilis; however 35 of these met the CSF criteria for a diagnosis supportive of neurosyphilis. Conclusions: The results of routine CSF analysis in patients with a possible diagnosis of neurosyphilis are inconsistently applied in the clinical setting, calling into question the value of routine CSF. Empirical neurosyphilis treatment should be considered up front in patients with high pre-test probability of the diagnosis. Keywords: Neurosyphilis, Rapid plasma reagin, Fluorescent treponemal antibody, Lumbar puncture, Cerebrospinal fluid

The introduction of the rotavirus vaccine, Rotarix, into the Fiji National Immunisation Program in 2012 has reduced the burden of rotavirus disease and hospitalisations in children less than 5 years of age. The aim of this study was to describe the pattern of rotavirus genotype diversity from 2005 to 2018, to investigate changes following the introduction of the rotavirus vaccine in Fiji. Faecal samples from children less than 5 years with acute diarrhoea between 2005 to 2018 were analysed at the WHO Rotavirus Regional Reference Laboratory at the Murdoch Children’s Research Institute, Melbourne, Australia, and positive samples were serotyped by EIA (2005–2006) or genotyped by heminested RT-PCR (2007 onwards). We observed a transient increase in the zoonotic strain equine-like G3P[8] in the initial period following vaccine introduction. G1P[8] and G2P[4], dominant genotypes prior to vaccine introduction, have not been detected since 2015 and 2014, respectively. A decrease in rotavirus genotypes G2P[8], G3P[6], G8P[8] and G9P[8] was also observed following vaccine introduction. Monitoring the rotavirus genotypes that cause diarrhoeal disease in children in Fiji is important to ensure that the rotavirus vaccine will continue to be protective and to enable early detection of new vaccine escape strains if this occurs.

Since 2018 The American Society of Transplant has recommended that Hepatitis C Virus seropositive positive, non-viremic donors (HCVAb+/NAT-) be considered non-infectious and safe for transplantation. This report describes clinical outcomes and HCV serological and virological outcomes following lung transplantation (LTx) utilizing such donors. This retrospective cohort study describes seven HCVAb+/NAT- donors used for bilateral LTx. Donor information was sourced from the national organ donation service and recipient information from the institutional LTx database. Seven deceased donors (three female, median age 53, range 37-63 years) acquired HCV from injecting drug use (n=4), blood products (n=1), unknown (n=1). Four donors had been previously treated and cleared of HCV (sustained virological response at 24 weeks) before death. Seven recipients (2 female, median age 56, range 51-71 years), with pulmonary fibrosis (n=4), re-do LTx (n=3), were waitlisted a median of 87 days (range 8-362). At post-LTx follow up, six of seven are alive with excellent graft function at a median 1625 days (range 779-2582). No patient developed clinical, biochemical or virological evidence of HCV infection, however five recipients demonstrated persistent HCVAb+ (all consistently NAT-) for a median 414 days (range 332-872) post-LTx. Two patients HCVAb sero-reverted at d731 and d1461. Two other recipients remained HCVAb- at d1621 and d1398 on repeat testing. There was no evidence of HCV transmission to seven LTx recipients from HCVAb+/NAT- life-saving lung donors, however persistent HCV Ab positivity was seen in 5 LTx recipients with two demonstrating remote sero-reversion. We postulate this may be a form of Passenger Lymphocyte Syndrome.

Background: In 2012, Fiji introduced the 10-valent pneumococcal conjugate vaccine (PCV10). We assessed the impact of PCV10 on invasive pneumococcal disease (IPD), probable bacterial or pneumococcal meningitis (PBPM), meningitis and sepsis 3-5 years post-introduction. Methods: Laboratory-confirmed IPD and PBPM cases were extracted from national laboratory records. ICD-10-AM coded all-cause meningitis and sepsis cases were extracted from national hospitalisation records. Incidence rate ratios were used to compare outcomes pre/post-PCV10, stratified by age groups: 1-23m, 2-4y, 5-9y, 10-19y, 20-54y, ≥55y. To account for different detection and serotyping methods in the pre-and post-PCV10 period, a Bayesian inference model estimated serotype-specific changes in IPD, using pneumococcal carriage and surveillance data. Findings: There were 423 IPD, 1,029 PBPM, 1,391 all-cause meningitis and 7,611 all-cause sepsis cases. Five years post-PCV10 introduction, IPD declined by 60% (95%CI: 37%, 76%) in children 1-23m months old, and in age groups 2-4y, 5-9y, 10-19y although confidence intervals spanned zero. PBPM declined by 36% (95%CI: 21%, 48%) among children 1-23 months old, and in all other age groups, although some confidence intervals spanned zero. Among children

Background: In 2012, Fiji became the first independent Pacific island country to introduce rotavirus vaccine. We describe the impact of rotavirus vaccine on all-cause diarrhoea admissions in all ages, and rotavirus diarrhoea in children

Scabies, impetigo, and other skin and soft tissue infections (SSTIs) are highly prevalent in many tropical, low-middle income settings, but information regarding their burden of disease is scarce. We conducted a surveillance of presentations of scabies and SSTIs, including impetigo, abscesses, cellulitis, and severe SSTI, to primary health facilities in Fiji. We established a monthly reporting system over the course of 50 weeks (July 2018–June 2019) for scabies and SSTIs at all 42 public primary health facilities in the Northern Division of Fiji (population, ≈131,914). For each case, information was collected regarding demographics, diagnosis, and treatment. There were 13,736 individual primary healthcare presentations with scabies, SSTI, or both (108.3 presentations per 1000 person-years; 95% confidence interval [CI], 106.6–110 presentations). The incidence was higher for males than for females (incidence rate ratio [IRR], 1.15; 95% CI, 1.11–1.19). Children younger than 5 years had the highest incidence among all age groups (339.1 per 1000 person-years). The incidence was higher among the iTaukei (indigenous) population (159.9 per 1000 person-years) compared with Fijians of Indian descent (30.1 per 1000 person-years; IRR, 5.32; 95% CI, 5.03–5.61). Abscesses had the highest incidence (63.5 per 1,000 person-years), followed by scabies (28.7 per 1,000 person-years) and impetigo (21.6 per 1,000 person-years). Scabies and SSTIs impose a substantial burden in Fiji and represent a high incidence of primary health presentations in this population. The incidence in low-middle income settings is up to 10-times higher than that in high-income settings. New public health strategies and further research are needed to address these conditions.

Antimicrobial resistance (AMR) is a critical global health threat with a disproportionate impact on low-income and middle-income countries (LMICs) due to their higher burden of infections, reduced laboratory surveillance infrastructure and fewer regulations governing antimicrobial use among humans or animals. While there have been increasing descriptions of AMR within many LMICs in WHO’s Western Pacific and South East Asian regions, there remains a paucity of data from Pacific Island countries and territories (PICTs). The PICTs represent 22 predominantly middle-income countries and territories with a combined population of 12 million people and 20 official languages, spread over hundreds of separate islands spanning an area corresponding to more than 15% of the earth’s surface. Our paper outlines the present state of the evidence regarding AMR in PICTs—discussing the present estimates of AMR and their accompanying limitations, important drivers of AMR, as well as outlining key priorities and potential solutions for tackling AMR in this region. Significant areas for action include developing National Action Plans, strengthening laboratory surveillance systems and educational activities targeted at both healthcare workers and the wider community. Ensuring adequate funding for AMR activities in PICTs is challenging given competing health and environmental priorities, in this context global or regional funding initiatives such as the Fleming Fund can play a key role.

Group A Streptococcus is a pathogen of global importance, but despite the ubiquity of group A Streptococcus infections, the relationship between infection, colonization, and immunity is still not completely understood. The M protein, encoded by the emm gene, is a major virulence factor and vaccine candidate and forms the basis of a number of classification systems. Longitudinal patterns of emm types collected from 457 Fijian schoolchildren over a 10-month period were analyzed. No evidence of tissue tropism was observed, and there was no apparent selective pressure or constraint of emm types. Patterns of emm type acquisition suggest limited, if any, modification of future infection based on infection history. Where impetigo is the dominant mode of transmission, circulating emm types either may not be constrained by ecological niches or population immunity to the M protein, or they may require several infections over a longer period of time to induce such immunity., info:eu-repo/semantics/published

Background Typhoid fever is endemic in Fiji. We sought to describe the epidemiology, clinical features and case fatality risk of blood culture-confirmed typhoid fever from January 2014 through December 2015. Methods Blood culture-positive patients were identified from a typhoid surveillance line list. A standardised case investigation form was used to record data from patients’ medical records. Results Of 542 patients, 518 (95.6%) were indigenous Fijians (iTaukei) and 285 (52.6%) were male. The median (IQR) age was 25 (16–38) y. Mean (SD) time from the onset of illness to admission was 11.1 (6.9) d. Of 365 patients with clinical information, 346 (96.9%) had fever, 239 (66.9%) diarrhoea, 113 (33.5%) vomiting, and 72 (30.2%) abdominal pain. There were 40 (11.0%) patients with complications, including 17 (4.7%) with shock, and 11 (3.0%) with hepatitis. Nine patients died for a case fatality risk of 1.7%. Of the 544 Salmonella Typhi isolates tested, none were resistant to first line antimicrobials; 3(0.8%) were resistant to ciprofloxacin and 5(1.4%) to nalidixic acid. Conclusions In Fiji, most blood culture-confirmed typhoid fever cases were in young adults. Common clinical manifestations were fever and gastrointestinal symptoms. Further studies are required to elucidate the factors associated with complications and death.

Objectives mcr-9.1 is a newly described mobile colistin resistance gene. We have noted its presence in multiple species of carbapenem-resistant Enterobacterales (CRE) from our institution. We aimed to determine the clinical features, genomic context and phenotypic impact of mcr-9.1 carriage in a series of patients between 2010 and 2019. Methods We identified 32 patients with mcr-9.1-carrying CRE isolates (mCRE) and collected demographic, antimicrobial exposure and infection data. Whole-genome sequencing (including short and long reads) was performed on 32 isolates. We assessed sequence similarity of mcr-9.1-harbouring plasmids, then compared our findings with plasmids for which sequence data were publicly available. Results There was no colistin exposure in patients prior to isolation of mCRE. mcr-9.1 was identified on IncHI2 plasmids across four different bacterial species and was co-located with blaIMP-4 in 23/30 plasmids studied. mCRE isolates did not demonstrate phenotypic colistin resistance, either at baseline or following sublethal colistin exposure, thus showing that mcr-9.1 alone is not sufficient for resistance. Publicly available sequence data indicated the presence of carbapenemase genes in 236/619 mcr-9.1-carrying genomes (38%). IncHI2 plasmids carrying mcr-9.1 and carbapenemase genes were detected in genomes from North America, Europe, North Africa, Asia and Oceania. Conclusions Spread of mcr-9.1 in CRE from our institution was driven by IncHI2 ‘superplasmids’, so termed because of their large size and their prolific carriage of resistance determinants. These were also detected in global CRE genomes. Phenotypic colistin resistance was not detected in our isolates but remains to be determined from global mCRE.

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.

Given the long term sequelae of untreated neurosyphilis and insensitive tests to detect treponemes in the cerebrospinal fluid, questions regarding the utility of a lumbar puncture and cerebrospinal fluid analysis either to confirm or exclude neurosyphilis are raised.

Proximal characteristics and conditions in the residential setting deserve greater attention for their potential to influence typhoid transmission. Using a case-control design in Central Division, Republic of Fiji, we examined bacterial (coliform and Escherichia coli) contamination and chemical composition of water and soil as potential vehicles of exposure to Salmonella Typhi, combining observational analysis of residential living conditions, geospatial analysis of household locations, and factor analysis to explore multivariate associations with the risk of developing typhoid fever. Factors positively associated with typhoid infection related to drainage [phosphate (OR 4.235, p = 0.042) and E. coli concentrations (OR 2.248, p = 0.029) in toilet drainage soil, housing [external condition (OR 3.712, p &lt, 0.001)], drinking water contamination (OR 2.732, p = 0.003) and sanitary condition (OR 1.973, p = 0.031). These five factors explained 42.5% of the cumulative variance and were significant in predicting typhoid infection. Our results support the hypothesis that a combination of spatial and biophysical attributes of the residential setting influence the probability of typhoid transmission, in this study, factors associated with poor drainage, flooding, and sanitary condition increase local exposure to contaminated water and soil, and thereby infection. These findings extend testing of causal assumptions beyond the immediate domestic domain, enhance the scope of traditional case control epidemiology and allow greater specificity of interventions at the scale of the residential setting.

Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule 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 were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Seasonal influenza like illness results in increased hospital presentations and unnecessary antibiotic use. Recent availability of rapid respiratory virus PCR testing allows rapid, accurate influenza diagnosis. A retrospective audit of turnaround time from sample collection to result availability after introduction of Xpert® Flu/RSV in a tertiary healthcare institution during a high incidence influenza season is described.

Two meticillin-resistant Staphylococcus aureus (MRSA) clones, sequence type (ST) 22 and ST239, have successfully spread globally. Across Australia, ST22 has supplanted ST239 as the main healthcare-associated MRSA. To understand the reasons underlying this shift, the epidemiology and clinical features of infections due to ST22 and ST239 MRSA isolates from a tertiary hospital in Melbourne, Australia were compared.Over six months, consecutive MRSA isolates with clinical data were collected from specimens referred to Alfred Health Pathology (AHP). Isolates were genotyped by a multi-locus-sequence-typing-based high-resolution melting method.Three hundred and twenty-eight of 1079 (30%) S. aureus isolated by AHP were MRSA. Of these, 313 were genotyped; 78 (25%) were clonal complex (CC) 22 (representing ST22) and 142 (45%) were CC239 (representing ST239). Common clinical syndromes included skin or soft tissue, respiratory tract and osteo-articular infections. On multi-variate logistic regression, compared with CC239, CC22 was associated with older patients [adjusted odds ratio (aOR) 1.04 for each year increase, 95% confidence interval (CI) 1.02-1.07)], and patients from subacute hospitals (aOR 2.7, 95% CI 1.2-5.8) or long-term care facilities (LTCFs; aOR 5.5, 95% CI 2.0-14.5). Median time from patient admission to MRSA isolation was nine days for CC239 and one day for CC22 (P 0.01). MRSA strain epidemiology varied according to hospital unit.CC22 and CC239 MRSA have differing ecological niches. CC22 is associated with elderly patients in LTCFs, and CC239 is associated with nosocomial acquisition. Infection control strategies involving LTCFs and their residents will likely be required to achieve continued MRSA control.

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., Author summary Klebsiellla pneumoniae is classified by the World Health Organization as a priority drug-resistant organism because it causes a significant burden of hospital infections that are extremely difficult to treat. However, outside of the hospital setting this bacterium is also an important cause of severe drug-susceptible infections. Until recently these two infection types were associated with distinct subsets of the K. pneumoniae population harbouring high prevalence of drug-resistance and virulence genes, respectively. However, there are now increasing reports of highly-virulent and difficult-to-treat K. pneumoniae strains that carry both resistance and virulence genes. In this study, we used genomic analyses to characterise and compare the evolutionary histories of drug-resistant and virulent K. pneumoniae. We show that the former are highly diverse, frequently acquiring novel genes through the processes of chromosomal recombination, plasmid and bacteriophage acquisition. In comparison, the latter show considerably lower gene content diversity, suggesting that they may be subject to some sort of limitation for gene acquisition. Consequently, we predict that drug-resistant K. pneumoniae are more likely to acquire virulence genes than virulent K. pneumoniae are to acquire drug-resistance genes.

BackgroundKlebsiella pneumoniaeis a recognised agent of multidrug-resistant (MDR) healthcare-associated infections, however individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulentK. pneumoniaeclones that cause severe community-associated infections such as liver abscess and pneumonia. Concerninglyiuchas also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in theK. pneumoniaepopulation.MethodsHere we examined the genetic diversity, distribution and mobilisation ofiucandiroloci among 2503K. pneumoniaegenomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance.ResultsIroandiucwere detected at low prevalence (iroand sixiuclineages that show distinct patterns of mobilisation and dissemination in theK. pneumoniaepopulation. The major burden ofiucandiroamongst the genomes analysed was due to two linked lineages (iuc1/iro1, 74% andiuc2/iro2, 14%), each carried by a distinct non-self-transmissible IncFIBKvirulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids ofK. pneumoniae. The otheriucandirolineages were associated with diverse plasmids, including some carrying FII conjugative transfer regions and some imported fromE. coli; the exceptions wereiro3(mobilised byICEKp1), andiuc4(fixed in the chromosome ofK. pneumoniaesubspeciesrhinoscleromatis).Iro/iucMGEs appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc), but were also detected at low prevalence in others such as MDR strain ST258.ConclusionsIucandiroare mobilised inK. pneumoniaevia a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDRK. pneumoniaestrains.

Mobile genetic elements (MGEs) that frequently transfer within and between bacterial species play a critical role in bacterial evolution, and often carry key accessory genes that associate with a bacteria’s ability to cause disease. MGEs carrying antimicrobial resistance (AMR) and/or virulence determinants are common in opportunistic pathogenKlebsiella pneumoniae, which are a leading cause of highly drug-resistant infections in hospitals. Well-characterised virulence determinants inK. pneumoniaeinclude the polyketide synthesis lociybtandclb(also known aspks), encoding the iron-scavenging siderophore yersiniabactin and genotoxin colibactin respectively. These loci are located within an MGE called ICEKp, which is the most common virulence-associated MGE ofK. pneumoniae,providing a mechanism for these virulence factors to spread within the population.Here we apply population genomics to investigate the prevalence, evolution and mobility ofybtandclbinK. pneumoniaepopulations through comparative analysis of 2,498 whole genome sequences. Theybtlocus was detected in 40% ofK. pneumoniaegenomes, particularly amongst those associated with invasive infections. We identified 17 distinctybtlineages and 3clblineages, each associated with one of 14 different structural variants of ICEKp. Comparison with the wider Enterobacteriaceae population showed occasional ICEKpacquisition by other members. Theclblocus was present in 14% of allK. pneumoniaeand 38.4% ofybt+ genomes. Hundreds of independent ICEKpintegration events were detected affecting hundreds of phylogenetically distinctK. pneumoniaelineages, including ≥19 in the globally-disseminated carbapenem-resistant clone CG258. A novel plasmid-encoded form ofybtwas also identified, representing a new mechanism forybtdispersal inK. pneumoniaepopulations. These data show that MGEs carryingybtandclbcirculate freely in theK. pneumoniaepopulation, including among multidrug-resistant strains, and should be considered a target for genomic surveillance along with AMR determinants.AUTHOR SUMMARYKlebsiella pneumoniaeinfections are becoming increasingly difficult to treat with antibiotics. SomeK. pneumoniaestrains also carry extra genes that allow them to synthesise yersiniabactin, an iron-scavenging molecule, which enhances their ability to cause disease. These genes are located on a genetic element that can easily transfer between strains. Here, we screened 2498K. pneumoniaegenome sequences and found substantial diversity in the yersiniabactin genes and the associated genetic elements, including a novel mechanism of transfer, and detected hundreds of distinct yersiniabactin acquisition events betweenK. pneumoniaestrains. We show that these yersiniabactin mobile genetic elements are specifically adapted to theK. pneumoniaepopulation but also occasionally acquired by other bacterial members belonging to the Enterobacteriaceae family such asE. coli.These insights into the movement and genetics of yersiniabactin genes allow tracking of the evolution and spread of yersiniabactin in globalK. pneumoniaepopulations and monitoring for acquisition of yersiniabactin in antibiotic-resistant strains.

In 2012, Fiji introduced rotavirus vaccine (Rotarix, GSK) into the national immunisation schedule. We describe the intussusception epidemiology prior to rotavirus vaccine, temporal association of intussusception cases to administration of rotavirus vaccine, and estimate the additional number of intussusception cases that may be associated with rotavirus vaccine. A retrospective review of intussusception cases for children aged

The first case of Shigella-associated acalculous cholecystitis is described. A 27-year-old woman presented to hospital with diarrhoea and acute acalculous cholecystitis one day after return to Australia from Vietnam. Her feces culture grew multi-drug resistant ESBL-producing Shigella sonnei and she improved with antimicrobial therapy and intravenous fluids.

A short-term program of performing serial active screening cultures (ASC) in the intensive care unit was instituted to establish a method for the detection of antibiotic-resistant Gram-negative bacteria (GNB) and the local rates of colonisation. Of all submitted ASC, 25.9% (30/116 collected swabs) isolated an antibiotic-resistant GNB. ChromID ESBL agar (bioMérieux, France) identified the majority of these organisms, with the additional antibiotic-impregnated media [MacConkey agar (MCA) with ciprofloxacin, MCA with gentamicin and MCA with ceftazidime] adding limited benefit. Compared to swabs performed on admission, 37.8% (14/37) of patients cultured a new antibiotic-resistant isolate on discharge. Serial screening in intensive care has the ability to identify patients with unrecognised colonisation with antibiotic-resistant GNB; however, the increase in the laboratory workload and logistical challenges in the collection of the surveillance swabs may limit this program's expansion.

BackgroundKlebsiella pneumoniae is a leading cause of extended-spectrum beta-lactamase (ESBL) producing hospital-associated infections, for which elderly patients are at increased risk.MethodsWe conducted a 1-year prospective cohort study, in which a third of patients admitted to two 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.ResultsK. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8% and ESBL carriage 1.7%. K. pneumoniae infection incidence 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-15and belonged to clones associated with hospital-acquired ESBL infections in other countries (ST29, ST323, 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.ConclusionsThe 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 control. Rectal screening for ESBL organisms upon admission to geriatric wards could help inform patient management and infection control in such facilities.SummaryPatients’ own gut microbiota were the major source of K. pneumoniae, but extended-spectrum beta-lactamase strains were acquired in the referring hospital. This highlights the potential for rectal screening, and the importance of the wider hospital network, for local risk management.

Background: Invasive and disseminated Mycoplasma hominis infections are well recognized but uncommon complications in solid organ transplant recipients. In a single center, a cluster of M. hominis infections were identified in lung transplant recipients from the same thoracic intensive care unit (ICU). We sought to determine the source(s) of these infections. Methods: Medical records of the donor and infected transplant recipients were reviewed for clinical characteristics. Clinical specimens underwent routine processing with subculture on Mycoplasma-specific Hayflick agar. Mycoplasma hominis identification was confirmed using sequencing of the 16S ribosomal RNA gene. Mycoplasma hominis isolates were subjected to whole-genome sequencing on the Illumina NextSeq platform. Results: Three lung transplant recipients presented with invasive M. hominis infections at multiple sites characterized by purulent infections without organisms detected by Gram staining. Each patient had a separate donor; however, pretransplant bronchoalveolar lavage fluid was only available from the donor for patient 1, which subsequently grew M. hominis. Phylo- and pangenomic analyses indicated that the isolates from the donor and the corresponding recipient (patient 1) were closely related and formed a distinct single clade. In contrast, isolates from patients 2 and 3 were unrelated and divergent from one another. Conclusions: Mycoplasma hominis should be considered a cause of donor-derived infection. Genomic data suggest donor-to-recipient transmission of M. hominis. Additional patients co-located in the ICU were found to have genetically unrelated M. hominis isolates, excluding patient-to-patient transmission.

As part of the World Health Organization Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance in Suva, Fiji, cerebrospinal fluid (CSF) samples from suspected meningitis patients of all ages were examined by traditional methods (culture, Gram stain, and latex agglutination for bacterial antigen) and qPCR for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Of 266 samples tested, pathogens were identified in 47 (17.7%). S. pneumoniae was the most common pathogen detected (n = 17) followed by N. meningitidis (n = 13). The use of qPCR significantly increased detection of IB-VPD pathogens (P = 0.0001): of 35 samples that were qPCR positive for S. pneumoniae, N. meningitidis, and H. influenzae, only 10 were culture positive. This was particularly relevant for N. meningitidis, as only 1/13 cases was culture positive. Molecular serotyping by microarray was used to determine pneumococcal serotypes from 9 of 16 (56%) of samples using DNA directly extracted from CSF specimens. Results indicate that qPCR significantly increases detection of S. pneumoniae, N. meningitidis, and H. influenzae in CSF, and that application of molecular diagnostics is a feasible way to enhance local and global surveillance for IB-VPD.

BackgroundKlebsiella pneumoniae is an opportunistic pathogen and a leading cause of hospital-associated (HA) infections. Patients in intensive care units (ICUs) are particularly at risk, and outbreaks are frequently reported in ICUs. K. pneumoniae is also part of the healthy human microbiome, providing a potential reservoir for HA infection. However, the frequency of K. pneumoniae gut colonization and its contribution to HA infections are not well characterized.MethodsWe conducted one-year prospective cohort study of ICU patients. Participants (n=498) were screened for rectal and throat carriage of K. pneumoniae shortly after admission, and clinical information was extracted from hospital records.K. pneumoniae isolated from screening swabs and clinical diagnostic samples were characterized using whole genome sequencing. Genomic and epidemiological data were combined to identify likely transmission events.Results and ConclusionsK. pneumoniae carriage frequencies were estimated at 6% (95% CI, 3%-8%) amongst ICU patients admitted direct from the community, and 19% (95% CI, 14% – 51%) amongst those who had recent contact with healthcare. Gut colonisation on admission was significantly associated with subsequent K. pneumoniae infection (infection risk 16% vs 3%, OR=6.9, pK. pneumoniae infections in ICU). In contrast, 49% of K. pneumoniae infections were caused by a strain that was unique to the patient, and 48% of patients with K. pneumoniae infections who participated in screening were positive for prior colonisation. These data confirm K. pneumoniae colonisation is a significant risk factor for subsequent infection in ICU, and indicate that half of all K. pneumoniae infections result from patients’ own microbiota. Screening for colonisation on admission could limit risk of infection in the colonised patient and others.

BackgroundKlebsiella pneumoniaeand close relatives are a growing cause of healthcare-associated infections for which increasing rates of multi-drug resistance are a major concern. TheKlebsiellapolysaccharide capsule is a major virulence determinant and epidemiological marker. However, little is known about capsule epidemiology since serological typing is not widely accessible, and many isolates are serologically non-typeable. Molecular methods for capsular typing are needed, but existing methods lack sensitivity and specificity and fail to take advantage of the information available in whole-genome sequence data, which is increasingly being generated for surveillance and investigation ofKlebsiella.MethodsWe investigated the diversity of capsule synthesis loci (K loci) among a large, diverse collection of 2503 genome sequences ofK. pneumoniaeand closely related species. We incorporated analyses of both full-length K locus DNA sequences and clustered protein coding sequences to identify, annotate and compare K locus structures, and we propose a novel method for identifying K loci based on full locus information extracted from whole genome sequences.ResultsA total of 134 distinct K loci were identified, including 31 novel types. Comparative analysis of K locus gene content detected 508 unique protein coding gene clusters that appear to reassort via homologous recombination, generating novel K locus types. Extensive nucleotide diversity was detected among thewziandwzcgenes, both within and between K loci, indicating that current typing schemes based on these genes are inadequate. As a solution, we introduceKaptive, a novel software tool that automates the process of identifying K loci from large sets ofKlebsiellagenomes based on full locus information.ConclusionsThis work highlights the extensive diversity ofKlebsiellaK loci and the proteins that they encode. We propose a standardised K locus nomenclature forKlebsiella, present a curated reference database of all known K loci, and introduce a tool for identifying K loci from genome data (https://github.com/katholt/Kaptive). These developments constitute important new resources for theKlebsiellacommunity for use in genomic surveillance and epidemiology.

Background Nocardia bacteremia is a rare but important phenomenon, with previous studies describing a 50% mortality rate. We undertake a single-center review and the largest systematic review of Nocardia bacteremia performed over the past 20 years. Methods A single-center review of cases of Nocardia bacteremia was performed using hospital microbiology records from January 1, 2010 to December 31, 2017. A systematic literature review was also performed to identify cases of Nocardia bacteremia described in the English language literature between January 1, 1999 and December 31, 2018 using the NCBI PubMed database and snowballing from citations of relevant publications. Results Single-center case series: Four cases of Nocardia bacteremia are described. Three patients had an intravascular device in situ prior to the onset of Nocardia bacteremia and three patients were immunocompromised; one patient had both risk factors. Systematic literature review: A systematic review identified 50 publications that described 85 cases with sufficient patient data to be reviewed in detail. Including the 4 cases described in our institution, 89 cases of Nocardia bacteremia were included in the analysis. The median age was 57 years [interquartile range (IQR) 42–68] and 69% were male. Eighty-two percent of cases were immunocompromised and 38% had endovascular devices. Pulmonary infection was the most common concurrent site of clinical disease (66%), followed by central nervous system (25%), pleural (17%) disease, and endocarditis (11%). Blood cultures were the only positive microbiological specimen that isolated Nocardia in 45% of cases. Median incubation time to blood culture positivity was 4 days [IQR 3–6]. Thirty-day all-cause mortality was 24% and overall all-cause mortality was 42%. Conclusion Four new cases of Nocardia bacteremia are described. Isolation of Nocardia from blood cultures is rare but represents serious infection with high associated overall mortality. Nocardia bacteremia is most frequently identified in immunocompromised patients and those with intravascular devices. Disclosures All authors: No reported disclosures.

OBJECTIVES Antimicrobial stewardship programs are recommended to reduce antimicrobial resistance by reducing inappropriate use of antimicrobials. We implemented an antimicrobial stewardship program and aimed to evaluate its effect on broad-spectrum antimicrobial use. DESIGN, SETTING AND PARTICIPANTS Observational study with historical control using interrupted time series analysis conducted in a tertiary referral hospital. Hospital inpatients prescribed restricted antimicrobials for non-standard indications, where approval had expired or without approval. INTERVENTION Baseline period of 30 months immediately followed by an 18-03 intervention period commencing January 2011. MAIN OUTCOME MEASURES Number and type of interventions made by antimicrobial stewardship team; monthly rate of use of broad-spectrum antimicrobial agents (in defined daily doses/1000 occupied bed-18s). RESULTS The antimicrobial stewardship team made 1104 recommendations in 779 patients during the 18-03 intervention period. In 64% of cases, the recommendation was made to cease or de-escalate the antimicrobial therapy, or to change from intravenous to oral therapy. The introduction of the intervention resulted in an immediate 17% (95% CI, 13%-20%) reduction in broad-spectrum antimicrobial use in the intensive care unit and a 10% (95% CI, 4%-16%) reduction in broad-spectrum antimicrobial use outside the intensive care unit. Reductions were particularly seen in cephalosporin and glycopeptide use, although these were partially offset by increases in the use of β-lactam-β-lactamase inhibitors. CONCLUSIONS The introduction of an antimicrobial stewardship program, including postprescription review, resulted in an immediate reduction in broad-spectrum antimicrobial use in a tertiary referral centre. However, the effect of this intervention reduced over time.