Microbiology & Immunology - Research Publications

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    Bioinformatic investigation of discordant sequence data for SARS- CoV-2: insights for robust genomic analysis during pandemic surveillance
    Zufan, SE ; Lau, KA ; Donald, A ; Hoang, T ; Foster, CSP ; Sikazwe, C ; Theis, T ; Rawlinson, WD ; Ballard, SA ; Stinear, TP ; Howden, BP ; Jennison, AV ; Seemann, T (MICROBIOLOGY SOC, 2023-11)
    The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants' bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.
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    High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks
    Zufan, SE ; Mercoulia, K ; Kwong, JC ; Judd, LM ; Howden, BP ; Seemann, T ; Stinear, TP ; Rantsiou, K (AMER SOC MICROBIOLOGY, 2024-01-11)
    This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.
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    Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.
    Carey, ME ; Dyson, ZA ; Ingle, DJ ; Amir, A ; Aworh, MK ; Chattaway, MA ; Chew, KL ; Crump, JA ; Feasey, NA ; Howden, BP ; Keddy, KH ; Maes, M ; Parry, CM ; Van Puyvelde, S ; Webb, HE ; Afolayan, AO ; Alexander, AP ; Anandan, S ; Andrews, JR ; Ashton, PM ; Basnyat, B ; Bavdekar, A ; Bogoch, II ; Clemens, JD ; da Silva, KE ; De, A ; de Ligt, J ; Diaz Guevara, PL ; Dolecek, C ; Dutta, S ; Ehlers, MM ; Francois Watkins, L ; Garrett, DO ; Godbole, G ; Gordon, MA ; Greenhill, AR ; Griffin, C ; Gupta, M ; Hendriksen, RS ; Heyderman, RS ; Hooda, Y ; Hormazabal, JC ; Ikhimiukor, OO ; Iqbal, J ; Jacob, JJ ; Jenkins, C ; Jinka, DR ; John, J ; Kang, G ; Kanteh, A ; Kapil, A ; Karkey, A ; Kariuki, S ; Kingsley, RA ; Koshy, RM ; Lauer, AC ; Levine, MM ; Lingegowda, RK ; Luby, SP ; Mackenzie, GA ; Mashe, T ; Msefula, C ; Mutreja, A ; Nagaraj, G ; Nagaraj, S ; Nair, S ; Naseri, TK ; Nimarota-Brown, S ; Njamkepo, E ; Okeke, IN ; Perumal, SPB ; Pollard, AJ ; Pragasam, AK ; Qadri, F ; Qamar, FN ; Rahman, SIA ; Rambocus, SD ; Rasko, DA ; Ray, P ; Robins-Browne, R ; Rongsen-Chandola, T ; Rutanga, JP ; Saha, SK ; Saha, S ; Saigal, K ; Sajib, MSI ; Seidman, JC ; Shakya, J ; Shamanna, V ; Shastri, J ; Shrestha, R ; Sia, S ; Sikorski, MJ ; Singh, A ; Smith, AM ; Tagg, KA ; Tamrakar, D ; Tanmoy, AM ; Thomas, M ; Thomas, MS ; Thomsen, R ; Thomson, NR ; Tupua, S ; Vaidya, K ; Valcanis, M ; Veeraraghavan, B ; Weill, F-X ; Wright, J ; Dougan, G ; Argimón, S ; Keane, JA ; Aanensen, DM ; Baker, S ; Holt, KE ; Global Typhoid Genomics Consortium Group Authorship, (eLife Sciences Publications, Ltd, 2023-09-12)
    BACKGROUND: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). METHODS: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. RESULTS: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. CONCLUSIONS: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. FUNDING: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).
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    The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus
    Sharkey, LKR ; Guerillot, R ; Walsh, CJ ; Turner, AM ; Lee, JYH ; Neville, SL ; Klatt, S ; Baines, SL ; Pidot, SJ ; Rossello, FJ ; Seemann, T ; McWilliam, HEG ; Cho, E ; Carter, GP ; Howden, BP ; McDevitt, CA ; Hachani, A ; Stinear, TP ; Monk, IR ; Torres, VJ (AMER SOC MICROBIOLOGY, 2023-12-19)
    Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.
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    Clinical Staphylococcus aureus inhibits human T-cell activity through interaction with the PD-1 receptor
    Mellergaard, M ; Skovbakke, SL ; Jepsen, SD ; Panagiotopoulou, N ; Hansen, ABR ; Tian, W ; Lund, A ; Hogh, RI ; Moller, SH ; Guerillot, R ; Hayes, AS ; Erikstrup, LT ; Andresen, L ; Peleg, AY ; Larsen, AR ; Stinear, TP ; Handberg, A ; Erikstrup, C ; Howden, BP ; Goletz, S ; Frees, D ; Skov, S ; Pier, GB ; Fey, PD (AMER SOC MICROBIOLOGY, 2023-10-06)
    Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.
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    Comparison of contemporary invasive and non-invasive Streptococcus pneumoniae isolates reveals new insights into circulating anti-microbial resistance determinants
    Higgs, C ; Kumar, LS ; Stevens, K ; Strachan, J ; Korman, T ; Horan, K ; Daniel, D ; Russell, M ; Mcdevitt, CA ; Sherry, NL ; Stinear, TP ; Howden, BP ; Gorrie, CL ; Tamma, PD (AMER SOC MICROBIOLOGY, 2023-10-12)
    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.
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    Population structure, serotype distribution and antibiotic resistance of Streptococcus pneumoniae causing invasive disease in Victoria, Australia
    Higgs, C ; Kumar, LS ; Stevens, K ; Strachan, J ; Sherry, NL ; Horan, K ; Zhang, J ; Stinear, TP ; Howden, BP ; Gorrie, CL (MICROBIOLOGY SOC, 2023-07)
    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.
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    Public health implementation of pathogen genomics: the role for accreditation and application of ISO standards
    Ballard, SA ; Sherry, NL ; Howden, BP (MICROBIOLOGY SOC, 2023-08)
    Pathogen genomics has transitioned rapidly from the research setting into a powerful tool now routinely used in public health microbiology, for surveillance, outbreak investigations and disease control. As these investigations can have significant public health, treatment and legal impacts, we must ensure the accuracy of these results through validation of testing processes. For laboratories working in this space, it is important to approach this work with a quality and accreditation framework in mind, working towards implementation of quality systems and test validation that meet international regulatory standards. Here we outline the key international standards and processes that lead toward accreditation for pathogen genomics.
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    Genomic surveillance for antimicrobial resistance - a One Health perspective
    Djordjevic, SP ; Jarocki, VM ; Seemann, T ; Cummins, ML ; Watt, AE ; Drigo, B ; Wyrsch, ER ; Reid, CJ ; Donner, E ; Howden, BP (NATURE PORTFOLIO, 2024-02)
    Antimicrobial resistance (AMR) - the ability of microorganisms to adapt and survive under diverse chemical selection pressures - is influenced by complex interactions between humans, companion and food-producing animals, wildlife, insects and the environment. To understand and manage the threat posed to health (human, animal, plant and environmental) and security (food and water security and biosecurity), a multifaceted 'One Health' approach to AMR surveillance is required. Genomic technologies have enabled monitoring of the mobilization, persistence and abundance of AMR genes and mutations within and between microbial populations. Their adoption has also allowed source-tracing of AMR pathogens and modelling of AMR evolution and transmission. Here, we highlight recent advances in genomic AMR surveillance and the relative strengths of different technologies for AMR surveillance and research. We showcase recent insights derived from One Health genomic surveillance and consider the challenges to broader adoption both in developed and in lower- and middle-income countries.
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    Detection of Streptococcus pyogenes M1UK in Australia and characterization of the mutation driving enhanced expression of superantigen SpeA
    Davies, MRR ; Keller, N ; Brouwer, S ; Jespersen, MGG ; Cork, AJJ ; Hayes, AJ ; Pitt, MEE ; De Oliveira, DMP ; Harbison-Price, N ; Bertolla, OMM ; Mediati, DGG ; Curren, BFF ; Taiaroa, G ; Lacey, JAA ; Smith, HVV ; Fang, N-X ; Coin, LJM ; Stevens, K ; Tong, SYC ; Sanderson-Smith, M ; Tree, JJJ ; Irwin, ADD ; Grimwood, K ; Howden, BPP ; Jennison, AVV ; Walker, MJJ (NATURE PORTFOLIO, 2023-02-24)
    A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.