Increase in invasive Haemophilus influenzae serotype A infections during the COVID-19 pandemic in New South Wales, Australia.

Haemophilus influenzae, a causative agent of severe invasive infections such as meningitis, sepsis and pneumonia, is classified into encapsulated or typeable (represented by serotypes A to F) and non-typeable varieties (NTHi) by the presence or absence of the polysaccharide capsule. Invasive disease caused by H. influenzae type B (HIB) can be prevented through vaccination which remains the main disease control intervention in many countries. This study examined the genomic diversity of circulating H. influenzae strains associated with invasive disease in New South Wales, Australia, before and during the COVID-19 pandemic. Ninety-six isolates representing 95 cases of invasive H. influenzae infections (iHi) diagnosed between January 2017 and September 2022 were typed and characterised using whole genome sequencing. These cases were caused by serotypes A (n=24), B (n=35), E (n=3), F (n=2) and NTHi (n=32). There was an apparent decline in the number of iHi infections during the COVID-19 pandemic, with a corresponding increase in the proportion of iHi cases caused by serotype A (HIA), which returned to pre-pandemic levels in 2022. Fifteen isolates associated with HIB or non-typeable iHi were resistant to ß-lactams due to a PBP3 mutation or carriage of blaTEM-1. Further, capsular gene duplication was observed in HIB isolates but was not found in HIA. These findings provide important baseline genomic data for ongoing iHi surveillance and control.

Prevalence of opportunistic pathogens and anti-microbial resistance in urban aquaculture ponds.

Bacterial antimicrobial resistance (AMR) has emerged as a significant concern worldwide. The microbial community profile and potential AMR level in aquaculture ponds are often undervalued and attract less attention than other aquatic environments. We used amplicon and metagenomic shotgun sequencing to study microbial communities and AMR in six freshwater polyculture ponds in rural and urban areas of Bangladesh. Amplicon sequencing revealed different community structures between rural and urban ponds, with urban ponds having a higher bacterial diversity and opportunistic pathogens including Streptococcus, Staphylococcus, and Corynebacterium. Despite proteobacterial dominance, Firmicutes was the most interactive in the community network, especially in the urban ponds. Metagenomes showed that drug resistance was the most common type of AMR found, while metal resistance was only observed in urban ponds. AMR and metal resistance genes were found mainly in beta and gamma-proteobacteria in urban ponds, while AMR was found primarily in alpha-proteobacteria in rural ponds. We identified potential pathogens with a high profile of AMR and metal resistance in urban aquaculture ponds. As these ponds provide a significant source of protein for humans, our results raise significant concerns for the environmental sustainability of this food source and the dissemination of AMR into the food chain.

Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.

Dynamics of the benthic and planktic microbiomes in a Planktothrix-dominated toxic cyanobacterial bloom in Australia.

Cyanobacterial blooms are a concerning issue that threaten ecosystems, ecology and animal health. Bloom frequency has increased tremendously in recent times due to pollution, eutrophication of waterways, climate change, and changes in microbial community dynamics within the aquatic environment. Information about the spatiotemporal variation in microbial communities that drive a cyanobacterial bloom is very limited. Here, we analysed the spatiotemporal diversity and composition of bacterial communities, with a focus on cyanobacteria, during the bloom phase in a natural reservoir in Eastern Australia using high throughput amplicon sequencing. Sampling points and season had no influence on the richness and evenness of microbial communities during the bloom period, however some compositional differences were apparent across the seasons. Cyanobacteria were highly abundant during summer and autumn compared to winter and spring. The dominant cyanobacterial taxa were Planktothrix, Cyanobium and Microcystis and were found to be significantly abundant during summer and autumn. While cyanobacterial abundance soared in summer (25.4 %), dominated by Planktothrix (12.2 %) and Cyanobium (8.0 %), the diversity was highest in autumn (24.9 %) and consisted of Planktothrix (7.8 %), Nodularia (5.3 %), Planktothricoides (4.6 %), Microcystis (3.5 %), and Cyanobium (2.3 %). The strongly correlated non-photosynthetic Gastranaerophilales found in the sediment and water, suggested vertical transmission from the animal gut through faeces. To our knowledge, this is the first report of Planktothrix-driven toxic cyanobacterial bloom in Australia. Our study expands current understanding of the spatiotemporal variation in bacterial communities during a cyanobacterial bloom and sheds light on setting future management strategies for its control.

Legionella pneumophila serogroup 1 infection associated with the use of an apartment building spa pool.

Background: Legionnaires' disease is a notifiable condition in New South Wales (NSW), Australia; clinicians and laboratories are required to report the disease to NSW Health. We describe the investigation of a sporadic case associated with the use of a communal spa pool in the case's apartment building complex and the use of whole genome sequencing to examine relatedness between clinical and environmental Legionella pneumophila serogroup 1 (Lp1) strains. Methods: In February 2018, a confirmed case of Lp1 infection was notified in a man in his 60s hospitalised with pneumonia. We asked the clinical team to obtain sputum in the event we found a potential source. The case described the use of the communal spa pool in his apartment building on two occasions during the putative exposure period. Environmental Health Officers from the Public Health Unit inspected the spa pool and found that the free chlorine level was well below the recommended concentration; a water sample was submitted for microbial analysis. Results: Lp1 was grown from the case's sputum and microbial analysis of the spa water sample found Lp1 at a concentration of 20 CFU/mL. The human and environmental isolates were subjected to whole genome sequencing and found to be highly genomically related. There was no other plausible environmental source of legionella. Conclusions: Whole genome sequencing of the clinical and environmental Lp1 isolates implicated a contaminated spa pool as the source of the case's exposure. This strongly supports the application of whole genome sequencing to the investigation of single cases of legionellosis. Communal spa pools in apartment buildings are not regulated in most Australian jurisdictions but must be considered to pose a potential legionella risk if improperly maintained.

Genomic and transcriptomic variation in Bordetella spp. following induction of erythromycin resistance.

BACKGROUND: The emergence of macrolide resistance in Bordetella pertussis, the causative agent of pertussis, due to mutations in the 23S rRNA gene has been recently recognized. However, resistance mechanisms to macrolides in Bordetella parapertussis and Bordetella holmesii remain unknown. OBJECTIVES: This study investigated genomic changes induced by in vitro exposure to erythromycin in these three main pathogens responsible for pertussis-like disease. METHODS: A set of 10 clinical and reference strains of B. pertussis, B. parapertussis and B. holmesii was exposed to erythromycin for 15 weeks or 30 subculture passages. Antibiotic pressure was achieved by growth on the selective media with erythromycin Etest strips or impregnated discs. Genome polymorphisms and transcriptomic profiles were examined by short- and long-read sequencing of passaged isolates. RESULTS: B. parapertussis and B. holmesii isolates developed significant in vitro resistance to erythromycin (MIC >256 mg/L) within 2 to 7 weeks and at 5 to 12 weeks, respectively. B. pertussis remained phenotypically susceptible to the antibiotic following 15 weeks of exposure, with the MIC between 0.032 to 0.38 mg/L. Genomic analysis revealed that B. holmesii developed resistance due to mutations in the 23S rRNA gene. The resistance mechanism in B. parapertussis was hypothesized as being due to upregulation of an efflux pump mechanism. CONCLUSIONS: These findings indicate that both B. holmesii and B. parapertussis can be more prone to induced resistance following exposure to treatment with erythromycin than B. pertussis. The surveillance of macrolide resistance in Bordetella isolates recovered from patients with pertussis, especially persistent disease, is warranted.

Global Phylogeny of Mycobacterium avium and Identification of Mutation Hotspots During Niche Adaptation.

Mycobacterium avium is separated into four subspecies: M. avium subspecies avium (MAA), M. avium subspecies silvaticum (MAS), M. avium subspecies hominissuis (MAH), and M. avium subspecies paratuberculosis (MAP). Understanding the mechanisms of host and tissue adaptation leading to their clinical significance is vital to reduce the economic, welfare, and public health concerns associated with diseases they may cause in humans and animals. Despite substantial phenotypic diversity, the subspecies nomenclature is controversial due to high genetic similarity. Consequently, a set of 1,230 M. avium genomes was used to generate a phylogeny, investigate SNP hotspots, and identify subspecies-specific genes. Phylogeny reiterated the findings from previous work and established that Mycobacterium avium is a species made up of one highly diverse subspecies, known as MAH, and at least two clonal pathogens, named MAA and MAP. Pan-genomes identified coding sequences unique to each subspecies, and in conjunction with a mapping approach, mutation hotspot regions were revealed compared to the reference genomes for MAA, MAH, and MAP. These subspecies-specific genes may serve as valuable biomarkers, providing a deeper understanding of genetic differences between M. avium subspecies and the virulence mechanisms of mycobacteria. Furthermore, SNP analysis demonstrated common regions between subspecies that have undergone extensive mutations during niche adaptation. The findings provide insights into host and tissue specificity of this genetically conserved but phenotypically diverse species, with the potential to provide new diagnostic targets and epidemiological and therapeutic advances.

2-Methylhopanoids in geographically distinct, arid biological soil crusts are primarily cyanobacterial in origin.

Microbial palaeontology is largely reliant on the interpretation of geologically stable biomarkers or molecular fossils. Biomolecules that are both specific to particular groups of organisms and stable on a geological scale are invaluable for tracing the emergence and diversification of lifeforms, particularly in cases where mineral fossils are lacking. 2-Methylhopanoids and their diagenic product, 2-methylhopanes, are highly abundant bacterial membrane lipids, recoverable from samples in excess of a billion years old. In this work we used degenerate PCR, targeting 2-methylhopanoid biosynthesis genes, and sequencing to show that the ability to produce these molecules in arid biological soil crusts from deserts in diverse geographical locations (Utah, USA, and the Pilbara, Australia) is largely confined to cyanobacteria. These data suggest that 2-methylhopanes can be used as a proxy for cyanobacterial presence within these environments, contributing to our understanding of the emergence of terrestrial life on Earth.

Microbial Genomics as a Catalyst for Targeted Antivirulence Therapeutics.

Virulence arresting drugs (VAD) are an expanding class of antimicrobial treatment that act to "disarm" rather than kill bacteria. Despite an increasing number of VAD being registered for clinical use, uptake is hampered by the lack of methods that can identify patients who are most likely to benefit from these new agents. The application of pathogen genomics can facilitate the rational utilization of advanced therapeutics for infectious diseases. The development of genomic assessment of VAD targets is essential to support the early stages of VAD diffusion into infectious disease management. Genomic identification and characterization of VAD targets in clinical isolates can augment antimicrobial stewardship and pharmacovigilance. Personalized genomics guided use of VAD will provide crucial policy guidance to regulating agencies, assist hospitals to optimize the use of these expensive medicines and create market opportunities for biotech companies and diagnostic laboratories.

Comparative Genomics of Mycobacterium avium Subspecies Paratuberculosis Sheep Strains.

Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic enteritis that causes major losses to the global livestock industry. Further, it has been associated with human Crohn's disease. Several strains of MAP have been identified, the two major groups being sheep strain MAP, which includes the Type I and Type III sub-lineages, and the cattle strain or Type II MAP lineage, of which bison strains are a sub-grouping. Major genotypic, phenotypic and pathogenic variations have been identified in prior comparisons, but the research has predominately focused on cattle strains of MAP. In countries where the sheep industries are more prevalent, however, such as Australia and New Zealand, ovine JD is a substantial burden. An information gap exists regarding the genomic differences between sheep strain sub-lineages and the relevance of Type I and Type III MAP in terms of epidemiology and/or pathogenicity. We therefore investigated sheep MAP isolates from Australia and New Zealand using whole genome sequencing. For additional context, sheep MAP genome datasets were downloaded from the Sequence Read Archive and GenBank. The final dataset contained 18 Type III and 16 Type I isolates and the K10 cattle strain MAP reference genome. Using a pan-genome approach, an updated global phylogeny for sheep MAP from de novo assemblies was produced. When rooted with the K10 cattle reference strain, two distinct clades representing the lineages were apparent. The Australian and New Zealand isolates formed a distinct sub-clade within the type I lineage, while the European type I isolates formed another less closely related group. Within the type III lineage, isolates appeared more genetically diverse and were from a greater number of continents. Querying of the pan-genome and verification using BLAST analysis revealed lineage-specific variations (n = 13) including genes responsible for metabolism and stress responses. The genetic differences identified may represent important epidemiological and virulence traits specific to sheep MAP. This knowledge will potentially contribute to improved vaccine development and control measures for these strains.

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.

Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome sequencing and agent-based modeling.

In January 2020, a novel betacoronavirus (family Coronaviridae), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the etiological agent of a cluster of pneumonia cases occurring in Wuhan City, Hubei Province, China1,2. The disease arising from SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), subsequently spread rapidly causing a worldwide pandemic. Here we examine the added value of near real-time genome sequencing of SARS-CoV-2 in a subpopulation of infected patients during the first 10 weeks of COVID-19 containment in Australia and compare findings from genomic surveillance with predictions of a computational agent-based model (ABM). Using the Australian census data, the ABM generates over 24 million software agents representing the population of Australia, each with demographic attributes of an anonymous individual. It then simulates transmission of the disease over time, spreading from specific infection sources, using contact rates of individuals within different social contexts. We report that the prospective sequencing of SARS-CoV-2 clarified the probable source of infection in cases where epidemiological links could not be determined, significantly decreased the proportion of COVID-19 cases with contentious links, documented genomically similar cases associated with concurrent transmission in several institutions and identified previously unsuspected links. Only a quarter of sequenced cases appeared to be locally acquired and were concordant with predictions from the ABM. These high-resolution genomic data are crucial to track cases with locally acquired COVID-19 and for timely recognition of independent importations once border restrictions are lifted and trade and travel resume.

Optimization of sample preparation for culture-independent sequencing of Bordetella pertussis.

Bordetella pertussis, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. B. pertussis is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600 000 deaths are prevented annually by pertussis vaccination, but B. pertussis infection was still responsible for over 63 000 deaths globally in 2013. The re-emergence of B. pertussis has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling B. pertussis. Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for B. pertussis rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of B. pertussis isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type B. pertussis using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of B. pertussis were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of B. pertussis to human DNA was determined by real-time PCR for ERV3 and IS481 (as markers of human and B. pertussis DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and B. pertussis sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target B. pertussis. However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target B. pertussis DNA. Sequencing read depth improved five-fold in reads to B. pertussis. Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of B. pertussis in the era of culture-independent testing.

Pertactin-Negative and Filamentous Hemagglutinin-Negative Bordetella pertussis, Australia, 2013-2017.

During the 2008-2012 pertussis epidemic in Australia, pertactin (Prn)-negative Bordetella pertussis emerged. We analyzed 78 isolates from the 2013-2017 epidemic and documented continued expansion of Prn-negative ptxP3 B. pertussis strains. We also detected a filamentous hemagglutinin-negative and Prn-negative B. pertussis isolate.

Genome-wide comparison of Corynebacterium diphtheriae isolates from Australia identifies differences in the Pan-genomes between respiratory and cutaneous strains.

BACKGROUND: Corynebacterium diphtheriae is the main etiological agent of diphtheria, a global disease causing life-threatening infections, particularly in infants and children. Vaccination with diphtheria toxoid protects against infection with potent toxin producing strains. However a growing number of apparently non-toxigenic but potentially invasive C. diphtheriae strains are identified in countries with low prevalence of diphtheria, raising key questions about genomic structures and population dynamics of the species. This study examined genomic diversity among 48 C. diphtheriae isolates collected in Australia over a 12-year period using whole genome sequencing. Phylogeny was determined using SNP-based mapping and genome wide analysis. RESULTS: C. diphtheriae sequence type (ST) 32, a non-toxigenic clone with evidence of enhanced virulence that has been also circulating in Europe, appears to be endemic in Australia. Isolates from temporospatially related patients displayed the same ST and similarity in their core genomes. The genome-wide analysis highlighted a role of pilins, adhesion factors and iron utilization in infections caused by non-toxigenic strains. CONCLUSIONS: The genomic diversity of toxigenic and non-toxigenic strains of C. diphtheriae in Australia suggests multiple sources of infection and colonisation. Genomic surveillance of co-circulating toxigenic and non-toxigenic C. diphtheriae offer new insights into the evolution and virulence of pathogenic clones and can inform targeted public health actions and policy. The genomes presented in this investigation will contribute to the global surveillance of C. diphtheriae both for the monitoring of antibiotic resistance genes and virulent strains such as those belonging to ST32.

Genome-wide analysis of Streptococcus pneumoniae serogroup 19 in the decade after the introduction of pneumococcal conjugate vaccines in Australia.

The decline in invasive pneumococcal disease (IPD), following the introduction of the 7-valent pneumococcal conjugate vaccination (PCV-7), was tempered by emergence of non-vaccine serotypes, particularly 19A. In Australia, three years after PCV-7 was replaced by PCV-13, containing 19A and 19F antigens, serogroup 19 was still a prominent cause of IPD in children under five. In this study we examined the evolution of serogroup 19 before and after introduction of paediatric vaccines in New South Wales (NSW), Australia. Genomes of 124 serogroup 19 IPD isolates collected before (2004) and after introduction of PCV-7 (2008) and PCV-13 (2014), from children under five in NSW, were analysed. Eleven core genome sequence clusters (cgSC) and 35 multilocus sequence types (ST) were identified. The majority (78/124) of the isolates belonged to four cgSCs: cgSC7 (ST199), cgSC11 (ST320), cgSC8 (ST63) and cgSC9 (ST2345). ST63 and ST2345 were exclusively serotype 19A and accounted for its predominantly intermediate penicillin resistance; these two clusters first appeared in 2008 and largely disappeared after introduction of PCV-13. Serogroup 19 was responsible for the highest proportion of vaccine failures in NSW. Relatively low immunogenicity of serogroup 19 antigens and Australia's three-dose vaccine schedule could affect the population dynamics of this serogroup.

Relentless spread and adaptation of non-typeable vanA vancomycin-resistant Enterococcus faecium: a genome-wide investigation.

Background: VRE are prevalent among patients in ICUs. Non-typeable vanA VRE, due to loss of one of the genes used for MLST (pstS), have increased in Australia, suggestive of a new, hospital-acquired lineage. Objectives: To understand the significance of this lineage and its transmission using WGS of strains isolated from patients in ICUs across New South Wales, Australia. Methods: A total of 240 Enterococcus faecium isolates collected between February and May 2016, and identified by conventional PCR as vanA positive, were sequenced. Isolates originated from 12 ICUs in New South Wales, grouped according to six local health districts, and represented both rectal screening swab (n = 229) and clinical (n = 11) isolates. Results: ST analysis revealed the absence of the pstS gene in 84.2% (202 of 240) of vanA isolates. Two different non-typeable STs were present based on different allelic backbone patterns. Loss of the pstS gene appeared to be the result of multiple recombination events across this region. Evidence for pstS-negative lineage spread across all six local health districts was observed suggestive of inter-hospital transmission. In addition, multiple outbreaks were detected, some of which were protracted and lasted for the duration of the study. Conclusions: These findings confirmed the evolution, emergence and dissemination of non-typeable vanA E. faecium. This study has highlighted the utility of WGS when attempting to describe accurately the hospital-based pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism.

Detection and incidence of Bordetella holmesii in respiratory specimens from patients with pertussis-like symptoms in New South Wales, Australia.

Bordetella pertussis, the aetiological agent of whooping cough is routinely diagnosed by polymerase chain reaction (PCR) directed at IS481, an insertion sequence target also found in Bordetella holmesii. Recent reports have suggested that B. holmesii infections can be misdiagnosed as pertussis, which can have a significant impact on public health surveillance. This study investigated the presence of B. holmesii in B. pertussis positive clinical samples, in order to determine the incidence of B. holmesii. Clinical cases of pertussis diagnosed by IS481-specific PCR between October 2008 and March 2016 in New South Wales were included. Bordetella holmesii was detected through the simultaneous amplification of IS481 and B. holmesii specific insertions sequence, hIS1001. A total of 46 of 802 patients were identified to be positive for B. holmesii rather than B. pertussis, suggesting an incidence rate of 6.5% in 2009, 16.8% in 2010, 7.6% during 2013 and 8.1% during 2015. Bordetella holmesii infections were diagnosed during and between pertussis epidemics, however cases of B. holmesii and B. pertussis co-infections were not found. The predominant age group of B. holmesii infection was 11-18 years old, which was significantly different to the mean age of B. pertussis infections (0-6 years, p = 0.023). These findings revealed that B. holmesii was co-circulating alongside the B. pertussis epidemic for seven years, hidden from view, as B. holmesii infections have been diagnosed as B. pertussis. Confirmatory testing of B. pertussis positive samples for the presence of B. holmesii, especially during pertussis epidemics, should improve the quality of laboratory diagnosis and laboratory surveillance for pertussis. The presence of B. holmesii in Australia highlights the importance of testing for this pathogen and ongoing molecular surveillance that can guide the control of whooping cough.

Advances in Clinical Sample Preparation for Identification and Characterization of Bacterial Pathogens Using Metagenomics.

Whole genome sequencing (WGS) plays an increasing role in communicable disease control through high-resolution outbreak tracing, laboratory surveillance and diagnostics. However, WGS has traditionally relied on microbial culture in order to obtain pathogen specific DNA for sequencing. This has severely limited the application of whole genome sequencing on pathogens with fastidious culturing requirements. In addition, the widespread adoption of culture-independent diagnostic tests has reduced availability of cultured isolates for confirmatory testing and surveillance. These recent developments have created demand for the implementation of techniques enabling direct sequencing of microbial genomes in clinical samples without having to culture an isolate. However, sequencing of specific organisms from clinical samples can be affected by high levels of contaminating DNA from the host and other commensal microorganisms. Several methods have been introduced for selective lysis of host cells and/or separate specific organisms from a clinical sample. This review examines the different approaches for sample preparation that have been used in diagnostic and public health laboratories for metagenomic sequencing.