CO‐MRSA Infections in Australia Cost $3.5B Per Annum

Introduction The health and economic burdens of community-onset methicillin resistant Staphylococcus aureus (CO-MRSA) infections are needed to inform policy, planning and evidence-based practice. We aimed to synthesise data from a range of public sources to generate the first estimate of the national incidence and cost of CO-MRSA infections. Methods Incidences of CO-MRSA skin and soft tissue (SSTI), lower respiratory tract (LRTI) and bloodstream (BSI) infections were calculated for regions of Australia using data from existing literature and correspondence with specialists. Simulations estimated costs using treatment models developed for children and adults in primary or tertiary care settings and including bed-stay, diagnostics, procedures, mortalities and loss of productivity. Results Annually, in Australia there were found to be 3702 CO-MRSA SSTIs, 559 CO-MRSA BSIs and 425 CO-MRSA LRTIs, occupying 147,000 bed-days, including 1600 bed-days in intensive care. Incidence ranged from 4 /100,000 person-years in Tasmania to 243 /100,000 person-years in central Australia. CO-MRSA cost $3.5b annually in Australia. The higher incidence of SSTIs resulted in costs greater than summing the costs of BSIs and LRTIs. The greatest cost was mortality. The cost to the health system was found to be$1.9b, with bed occupancies accounting for ≥94%. Conclusion This was the first evaluation of the health and economic burden of CO-MRSA in Australia. We found a need for increased and more consistent data collection for a significant and expensive disease. Disclosure of Interest Statement: This research was funded by NHMRC grant GNT1027589

The Utility of High-Resolution Melting Analysis of SNP Nucleated PCR Amplicons—An MLST Based Staphylococcus aureus Typing Scheme

High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpson's Index of Diversity. Only G↔A, G↔T, C↔A, C↔T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 “melt types” (MelTs), and provides a Simpson's Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs

Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354–1363) to estimate divergence and effective reproduction numbers (Re) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth–death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (Re > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG

Sequence element enrichment analysis to determine the genetic basis of bacterial phenotypes

Peer reviewe

Phylodynamic signatures in the emergence of community-associated MRSA

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers

A Very Early-Branching Staphylococcus aureus Lineage Lacking the Carotenoid Pigment Staphyloxanthin

Here we discuss the evolution of the northern Australian Staphylococcus aureus isolate MSHR1132 genome. MSHR1132 belongs to the divergent clonal complex 75 lineage. The average nucleotide divergence between orthologous genes in MSHR1132 and typical S. aureus is approximately sevenfold greater than the maximum divergence observed in this species to date. MSHR1132 has a small accessory genome, which includes the well-characterized genomic islands, νSAα and νSaβ, suggesting that these elements were acquired well before the expansion of the typical S. aureus population. Other mobile elements show mosaic structure (the prophage φSa3) or evidence of recent acquisition from a typical S. aureus lineage (SCCmec, ICE6013 and plasmid pMSHR1132). There are two differences in gene repertoire compared with typical S. aureus that may be significant clues as to the genetic basis underlying the successful emergence of S. aureus as a pathogen. First, MSHR1132 lacks the genes for production of staphyloxanthin, the carotenoid pigment that confers upon S. aureus its characteristic golden color and protects against oxidative stress. The lack of pigment was demonstrated in 126 of 126 CC75 isolates. Second, a mobile clustered regularly interspaced short palindromic repeat (CRISPR) element is inserted into orfX of MSHR1132. Although common in other staphylococcal species, these elements are very rare within S. aureus and may impact accessory genome acquisition. The CRISPR spacer sequences reveal a history of attempted invasion by known S. aureus mobile elements. There is a case for the creation of a new taxon to accommodate this and related isolates

Streptococcus gallolyticus subsp. pasteurianus meningitis complicated by venous sinus thrombosis: A case report

A case of Streptococcus gallolyticus subsp. pasteurianus meningitis, unusually occurring in a splenectomized patient and complicated by cerebral venous thrombosis, is described. Following presentation with meningism and diagnosis and management of S. gallolyticus meningitis, the patient presented again with a further 4 days of fevers and subsequently developed left-sided paresthesias. Cerebral imaging revealed a venous thrombus in the right frontal cortical veins and left sigmoid sinus. The patient recovered following 4 weeks of intravenous ceftriaxone and anticoagulation with enoxaparin and then warfarin. Apart from the splenectomy, no underlying cause was found. The patient was commenced on life-long prophylactic amoxicillin, given appropriate vaccinations, and anticoagulated with warfarin. After initial difficulties, identification of the causative organism to the subspecies level was confirmed by analysis of short-read whole genome sequencing data. This case demonstrates two features that have not previously been reported for S. gallolyticus subsp. pasteurianus infections: splenectomy as a potential risk factor and that infection may be complicated by cerebral venous thrombosis. The resolution provided by whole genome sequencing was valuable in accurately identifying the bacterial subspecies

A Scenario-Based Survey of Expert Echocardiography Recommendations for Patients With Staphylococcus aureus Bacteremia at Varying Risk for Endocarditis

Importance: Echocardiography to detect infective endocarditis is regarded as a key quality indicator in the care of patients with Staphylococcus aureus bacteremia, but its application varies markedly between reported series. Understanding the reasons for this variation in practice is important to improve the use of this investigation. Objective: To identify expert clinicians' preferred echocardiography strategy for a variety of S aureus bacteremia scenarios in a hypothetical setting free from extrinsic constraints. Design, Setting, and Participants: Anonymous web-based survey study comprising 50 text-based scenarios describing patients with S aureus bacteremia and various combinations of risk factors for endocarditis. Other variables included patient age and the presence of an extracardiac focus of infection warranting prolonged treatment. The survey was emailed to participants between September 2018 and March 2019. Each respondent was asked to recommend 1 of 6 echocardiography strategies for up to 8 randomly selected scenarios. Respondents were primarily infectious diseases physicians, and more than half reported an annual caseload of more than 20 cases of S aureus bacteremia. Main Outcomes and Measures: The proportion of respondents selecting each of the 6 echocardiography strategies was calculated alongside Wilson score confidence intervals. Modified Fleiss κ statistics were used to described interrespondent variability. Generalized estimating equations were used to assess the associations between respondent- and scenario-level variables and the recommendation of an echocardiography strategy with a low negative likelihood ratio for infective endocarditis (ie, a highly exclusionary strategy). Results: A total of 656 respondents from 24 countries provided 4837 echocardiography recommendations across the 50 scenarios. Echocardiography recommendations were associated with scenarios' burden of endocarditis risk (multivariate odds ratio per point of the VIRSTA score, 1.4; 95% CI, 1.4-1.5; P < .001). Poor interrespondent agreement was seen across all scenarios (modified Fleiss κ, 0.06; 95% CI, 0.05-0.07) but was most notable for scenarios with a lower risk of endocarditis (modified Fleiss κ, 0.04; 95% CI, 0.03-0.05). The presence of an extracardiac focus of infection was also associated with the choice of echocardiography strategy (odds ratio for highly exclusionary strategy, 0.51; 95% CI, 0.45-0.58). Respondent location in continental Europe was associated with recommendations in favor of a highly exclusionary strategy (odds ratio, 1.8; 95% CI, 1.3-2.5) compared with location in Australia or New Zealand. Conclusions and Relevance: In this study, expert clinicians demonstrated active stratification by risk of endocarditis when making echocardiography recommendations for hypothetical patients with S aureus bacteremia. Substantial disagreement existed as to whether patients at lower risk of endocarditis should undergo transesophageal echocardiography-based echocardiography strategies