Reversible vancomycin susceptibility within emerging ST1421 Enterococcus faecium strains is associated with rearranged vanA-gene clusters and increased vanA plasmid copy number.

Vancomycin variable enterococci (VVE) are van-positive enterococci with a vancomycin-susceptible phenotype (VVE-S) that can convert to a resistant phenotype (VVE-R) and be selected for during vancomycin exposure. VVE-R outbreaks have been reported in Canada and Scandinavian countries. The aim of this study was to examine the presence of VVE in whole genome sequenced (WGS) Australian bacteremia Enterococcus faecium (Efm) isolates collected through the Australian Group on Antimicrobial resistance (AGAR) network. Eight potential VVEAus isolates, all identified as Efm ST1421, were selected based on the presence of vanA and a vancomycin-susceptible phenotype. During vancomycin selection, two potential VVE-S harboring intact vanHAX genes, but lacking the prototypic vanRS and vanZ genes, reverted to a resistant phenotype (VVEAus-R). Spontaneous VVEAus-R reversion occurred at a frequency of 4-6 × 10-8 resistant colonies per parent cell in vitro after 48 h and led to high-level vancomycin and teicoplanin resistance. The S to R reversion was associated with a 44-bp deletion in the vanHAX promoter region and an increased vanA plasmid copy number. The deletion in the vanHAX promoter region enables an alternative constitutive promoter for the expression of vanHAX. Acquisition of vancomycin resistance was associated with a low fitness cost compared with the corresponding VVEAus-S isolate. The relative proportion of VVEAus-R vs. VVEAus-S decreased over time in serial passages without vancomycin selection. Efm ST1421 is one of the predominant VanA-Efm multilocus sequence types found across most regions of Australia, and has also been associated with a major prolonged VVE outbreak in Danish hospitals.

Future-proofing and maximizing the utility of metadata: The PHA4GE SARS-CoV-2 contextual data specification package.

BACKGROUND: The Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) is a global coalition that is actively working to establish consensus standards, document and share best practices, improve the availability of critical bioinformatics tools and resources, and advocate for greater openness, interoperability, accessibility, and reproducibility in public health microbial bioinformatics. In the face of the current pandemic, PHA4GE has identified a need for a fit-for-purpose, open-source SARS-CoV-2 contextual data standard. RESULTS: As such, we have developed a SARS-CoV-2 contextual data specification package based on harmonizable, publicly available community standards. The specification can be implemented via a collection template, as well as an array of protocols and tools to support both the harmonization and submission of sequence data and contextual information to public biorepositories. CONCLUSIONS: Well-structured, rich contextual data add value, promote reuse, and enable aggregation and integration of disparate datasets. Adoption of the proposed standard and practices will better enable interoperability between datasets and systems, improve the consistency and utility of generated data, and ultimately facilitate novel insights and discoveries in SARS-CoV-2 and COVID-19. The package is now supported by the NCBI's BioSample database.

An implementation science approach to evaluating pathogen whole genome sequencing in public health.

BACKGROUND: Pathogen whole genome sequencing (WGS) is being incorporated into public health surveillance and disease control systems worldwide and has the potential to make significant contributions to infectious disease surveillance, outbreak investigation and infection prevention and control. However, to date, there are limited data regarding (i) the optimal models for integration of genomic data into epidemiological investigations and (ii) how to quantify and evaluate public health impacts resulting from genomic epidemiological investigations. METHODS: We developed the Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework to guide examination of the use of WGS in public health surveillance and disease control. We illustrate the use of this framework with three pathogens as case studies: Listeria monocytogenes, Mycobacterium tuberculosis and SARS-CoV-2. RESULTS: The framework utilises an adaptable whole-of-system approach towards understanding how interconnected elements in the public health application of pathogen genomics contribute to public health processes and outcomes. The three phases of the PG-PHASE Framework are designed to support understanding of WGS laboratory processes, analysis, reporting and data sharing, and how genomic data are utilised in public health practice across all stages, from the decision to send an isolate or sample for sequencing to the use of sequence data in public health surveillance, investigation and decision-making. Importantly, the phases can be used separately or in conjunction, depending on the need of the evaluator. Subsequent to conducting evaluation underpinned by the framework, avenues may be developed for strategic investment or interventions to improve utilisation of whole genome sequencing. CONCLUSIONS: Comprehensive evaluation is critical to support health departments, public health laboratories and other stakeholders to successfully incorporate microbial genomics into public health practice. The PG-PHASE Framework aims to assist public health laboratories, health departments and authorities who are either considering transitioning to whole genome sequencing or intending to assess the integration of WGS in public health practice, including the capacity to detect and respond to outbreaks and associated costs, challenges and facilitators in the utilisation of microbial genomics and public health impacts.

Key parameters for genomics-based real-time detection and tracking of multidrug-resistant bacteria: a systematic analysis.

BACKGROUND: Pairwise single nucleotide polymorphisms (SNPs) are a cornerstone of genomic approaches to the inference of transmission of multidrug-resistant (MDR) organisms in hospitals. However, the impact of many key analytical approaches on these inferences has not yet been systematically assessed. This study aims to make such a systematic assessment. METHODS: We conducted a 15-month prospective study (2-month pilot phase, 13-month implementation phase), across four hospital networks including eight hospitals in Melbourne, VIC, Australia. Patient clinical and screening samples containing one or more isolates of meticillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae were collected and underwent whole genome sequencing. Using the genome data from the top four most numerous sequence types from each species, 16 in total, we systematically assessed the: (1) impact of sample and reference genome diversity through multiple core genome alignments using different data subsets and reference genomes, (2) effect of masking of prophage and regions of recombination in the core genome alignments by assessing SNP distances before and after masking, (3) differences between a cumulative versus a 3-month sliding-window approach to sample genome inclusion in the dataset over time, and (4) the comparative effects each of these approaches had when applying a previously defined SNP threshold for inferring likely transmission. FINDINGS: 2275 samples were collected (397 during the pilot phase from April 4 to June 18, 2017; 1878 during the implementation phase from Oct 30, 2017, to Nov 30, 2018) from 1870 patients. Of these 2275 samples, 1537 were identified as arising from the four most numerous sequence types from each of the four target species of MDR organisms in this dataset (16 sequence types in total: S aureus ST5, ST22, ST45, and ST93; E faecium ST80, ST203, ST1421, and ST1424; K pneumoniae ST15, ST17, ST307, and ST323; and E coli ST38, ST131, ST648, and ST1193). Across the species, using a reference genome of the same sequence type provided a greater degree of pairwise SNP resolution, compared with species and outgroup-reference alignments that mostly resulted in inflated SNP distances and the possibility of missed transmission events. Omitting prophage regions had minimal effect; however, omitting recombination regions had a highly variable effect, often inflating the number of closely related pairs. Estimated SNP distances between isolate pairs over time were more consistent using a sliding-window than a cumulative approach. INTERPRETATION: We propose that the use of a closely related reference genome, without masking of prophage or recombination regions, and of a sliding-window approach for isolate inclusion is best for accurate and consistent MDR organism transmission inference, when using core genome alignments and SNP thresholds. These approaches provide increased stability and resolution, so SNP thresholds can be more reliably applied for putative transmission inference among diverse MDR organisms, reducing the chance of incorrectly inferring the presence or absence of close genetic relatedness and, therefore, transmission. The establishment of a broadly applicable and standardised approach, as proposed here, is necessary to implement widespread prospective genomic surveillance for MDR organism transmission. FUNDING: Melbourne Genomics Health Alliance, and National Health and Medical Research Council of Australia.

Complete microbial genomes for public health in Australia and the Southwest Pacific.

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.

Prolonged Outbreak of Multidrug-Resistant Shigella sonnei Harboring blaCTX-M-27 in Victoria, Australia.

In Australia, cases of shigellosis usually occur in returned travelers from regions of shigellosis endemicity or in men who have sex with men. Resistance to multiple antibiotics has significantly increased in Shigella sonnei isolates and represents a significant public health concern. We investigate an outbreak of multidrug-resistant S. sonnei in Victoria, Australia. We undertook whole-genome sequencing of 54 extended-spectrum-beta-lactamase (ESBL)-producing S. sonnei isolates received at the Microbiological Diagnostic Unit Public Health Laboratory between January 2019 and March 2020. The population structure and antimicrobial resistance profiles were identified by genomic analyses, with 73 previously characterized Australian S. sonnei isolates providing context. Epidemiological data, including age and sex of the shigellosis cases, were also collected. There was a significant increase in cases of ESBL S. sonnei from July 2019. Most of the ESBL S. sonnei isolates (65%) fell within a single cluster that was predominantly comprised of male cases that were characterized by the presence of the blaCTX-M-27 gene conferring resistance to extended-spectrum cephalosporins. These isolates were also multidrug resistant, including resistance to azithromycin and co-trimoxazole and reduced susceptibility to ciprofloxacin. Our data uncovered a prolonged clonal outbreak of ESBL S. sonnei infection that was likely first introduced by returned travelers and has subsequently been circulating locally in Australia. The emergence of a local outbreak of ESBL S. sonnei with a multidrug-resistant profile, including reduced susceptibility to ciprofloxacin, represents a significant public health threat.

Genomic Analysis of Multiresistant Staphylococcus capitis Associated with Neonatal Sepsis.

Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen.

Measuring Natural Selection.

In this chapter, I review the basic algorithm underlying the CODEML model implemented in the software package PAML. This is intended as a companion to the software's manual, and a primer to the extensive literature available on CODEML. At the end of this chapter, I hope that you will be able to understand enough of how CODEML operates to plan your own analyses.

Genomic Insights into a Sustained National Outbreak of Yersinia pseudotuberculosis.

In 2014, a sustained outbreak of yersiniosis due to Yersinia pseudotuberculosis occurred across all major cities in New Zealand (NZ), with a total of 220 laboratory-confirmed cases, representing one of the largest ever reported outbreaks of Y. pseudotuberculosis. Here, we performed whole genome sequencing of outbreak-associated isolates to produce the largest population analysis to date of Y. pseudotuberculosis, giving us unprecedented capacity to understand the emergence and evolution of the outbreak clone. Multivariate analysis incorporating our genomic and clinical epidemiological data strongly suggested a single point-source contamination of the food chain, with subsequent nationwide distribution of contaminated produce. We additionally uncovered significant diversity in key determinants of virulence, which we speculate may help explain the high morbidity linked to this outbreak.

Population history, phylogeography, and conservation genetics of the last Neotropical mega-herbivore, the lowland tapir (Tapirus terrestris).

BACKGROUND: Understanding the forces that shaped Neotropical diversity is central issue to explain tropical biodiversity and inform conservation action; yet few studies have examined large, widespread species. Lowland tapir (Tapirus terrrestris, Perissodactyla, Tapiridae) is the largest Neotropical herbivore whose ancestors arrived in South America during the Great American Biotic Interchange. A Pleistocene diversification is inferred for the genus Tapirus from the fossil record, but only two species survived the Pleistocene megafauna extinction. Here, we investigate the history of lowland tapir as revealed by variation at the mitochondrial gene Cytochrome b, compare it to the fossil data, and explore mechanisms that could have shaped the observed structure of current populations. RESULTS: Separate methodological approaches found mutually exclusive divergence times for lowland tapir, either in the late or in the early Pleistocene, although a late Pleistocene divergence is more in tune with the fossil record. Bayesian analysis favored mountain tapir (T. pinchaque) paraphyly in relation to lowland tapir over reciprocal monophyly, corroborating the inferences from the fossil data these species are sister taxa. A coalescent-based analysis rejected a null hypothesis of allopatric divergence, suggesting a complex history. Based on the geographic distribution of haplotypes we propose (i) a central role for western Amazonia in tapir diversification, with a key role of the ecological gradient along the transition between Andean subcloud forests and Amazon lowland forest, and (ii) that the Amazon river acted as an barrier to gene flow. Finally, the branching patterns and estimates based on nucleotide diversity indicate a population expansion after the Last Glacial Maximum. CONCLUSIONS: This study is the first examining lowland tapir phylogeography. Climatic events at the end of the Pleistocene, parapatric speciation, divergence along the Andean foothill, and role of the Amazon river, have similarly shaped the history of other taxa. Nevertheless further work with additional samples and loci is needed to improve our initial assessment. From a conservation perspective, we did not find a correspondence between genetic structure in lowland tapir and ecogeographic regions proposed to define conservation priorities in the Neotropics. This discrepancy sheds doubt into this scheme's ability to generate effective conservation planning for vagile species.