Extended water stagnation in buildings during the COVID-19 pandemic increases the risks posed by opportunistic pathogens.

The regrowth and subsequent exposure of opportunistic pathogens (OPs) whilst reopening buildings that have been locked down due to the stay-at-home restrictions to limit the spread of COVID-19, is a public health concern. To better understand such microbiological risks due to lowered occupancy and water demand in buildings, first and post-flush water samples (n = 48) were sampled from 24 drinking water outlets from eight university buildings in two campuses (urban and rural), with various end-user occupancies. Both campuses were served with chlorinated water originating from a single drinking water distribution system in South-East Queensland, situated 14 km apart, where the rural campus had lower chlorine residuals. Culture-dependent and culture-independent methods (such as flow cytometry, qPCR and 16S rRNA gene amplicon sequencing) were used concurrently to comprehensively characterise the OPs of interest (Legionella spp., Pseudomonas aeruginosa, and nontuberculous mycobacteria (NTM)) and the premise plumbing microbiome. Results showed that buildings with extended levels of stagnation had higher and diverse levels of microbial growth, as observed in taxonomic structure and composition of the microbial communities. NTM were ubiquitous in all the outlets sampled, regardless of campus or end-user occupancy of the buildings. qPCR and culture demonstrated prevalent and higher concentrations of NTM in buildings (averaging 3.25 log10[estimated genomic copies/mL]) with extended stagnation in the urban campus. Furthermore, flushing the outlets for 30 minutes restored residual and total chlorine, and subsequently decreased the levels of Legionella by a reduction of 1 log. However, this approach was insufficient to restore total and residual chlorine levels for the outlets in the rural campus, where both Legionella and NTM levels detected by qPCR remained unchanged, regardless of building occupancy. Our findings highlight that regular monitoring of operational parameters such as residual chlorine levels, and the implementation of water risk management plans are important for non-healthcare public buildings, as the levels of OPs in these environments are typically not assessed.

The effectiveness and role of phages in the disruption and inactivation of clinical P. aeruginosa biofilms.

Biofilms of P. aeruginosa are known to be resilient forms of survival of this opportunistic pathogen, both within the host and in natural or engineered environments. This study investigated the role of phages in the disruption and inactivation of clinical P. aeruginosa biofilms by previously isolated phages. All seven tested clinical strains formed biofilms in 56-80 h. Four previously isolated phages were effective in disrupting the formed biofilms when applied at multiplicity of infection (MOI) of 10, where phage cocktails had equivalent or worse performance than single phages. Phage treatments reduced the biofilms' biomass (cells and extracellular matrix) by 57.6-88.5% after 72 h of incubation. Biofilm disruption led to the detachment of 74.5-80.4% of the cells. The phages were also able to kill the cells from the biofilms, reducing the living cell counts by approximately 40.5-62.0% after a single treatment. A fraction of 24-80% of these killed cells were also lysed due to phage action. This study showed that phages can have a relevant role in disrupting, inactivating, and destroying P. aeruginosa biofilms, which can be used in the development of treatment processes to complement or replace antibiotics and/or disinfectants.

A bacterial genome assembly and annotation laboratory using a virtual machine.

With the global increase of infections caused by antibiotic-resistant bacterial strains, there is an urgent need for new methods of tackling the issue. Genomic analysis of bacterial strains can help to understand their virulence and antibiotic resistance profile. Bioinformatic skills are in great demand across the biological sciences. We designed a workshop that allows university students to learn the process of genome assembly using command-line tools within a virtual machine on a Linux operating system. We use Illumina and Nanopore short and long-read raw sequences to reveal the advantages and disadvantages of short, long, and hybrid assembly methods. The workshop teaches how to assess read and assembly quality, perform genome annotation, and analyze pathogenicity, antibiotic and phage resistance. The workshop is intended for a five-week teaching period and is concluded by a student poster presentation assessment.

Three synthetic biology applications and their paths to impact in Australia: Cane toads, bacteriophages, and biomining microbes.

Synthetic biology [synbio] applications have the potential to assist in addressing significant global health and environmental challenges. Australian research institutes are investing in formative research to develop synbio technologies capable of meeting these challenges. Alongside the laboratory research, investigating the broader social, institutional, and ethical considerations that synbio presents has been a priority. We conducted targeted qualitative research to uncover the barriers and opportunities for a range of multisectoral stakeholders identified as potential end-users of the science under development. The research provides insights into the research implementation environment for three synthetic biology applications: (1) gene editing cane toads (Rhinella marina) to reduce their environmental impact; (2) engineering bacteriophages to combat antimicrobial resistance in humans; and (3) engineering microbes to improve biomining efficiency in the mining industry. In-depth interviews (N = 23) with government, research and civil society representatives revealed key challenges in the impact pathway for each application. The strongest themes uncovered during interviews related to perceived negative public attitudes towards genetic technologies, a lack of investment in critical research infrastructure, unclear regulatory pathways and the presence of a strong social and environmental imperative underpinning technology development. These findings reveal specific entry points for further engagement with the most immediate end-users of synbio. Separate from research on public attitudes to synbio, the cases highlight the various hurdles to achieving research impact, according to experts who will likely use, approve or invest in these applications in the future. The themes uncovered inform avenues for strengthening engagement and research coordination in Australia and elsewhere.

Novel Bacteriophages Show Activity against Selected Australian Clinical Strains of Pseudomonas aeruginosa.

Multi-drug resistant (MDR) clinical strains of Pseudomonas aeruginosa are the most prevalent bacteria in the lungs of patients with cystic fibrosis (CF) and burn wounds and among the most common in immunocompromised hospital patients in Australia. There are currently no promising antibiotics in the pipeline being developed against these strains. Phage therapy, which uses viruses known as bacteriophages to infect and kill pathogenic bacteria, could be a possible alternative treatment. To this end, we isolated and characterised four novel phages against Australian clinical strains of P. aeruginosa isolated from patients with cystic fibrosis, from infected blood and joint aspirate in Southeast Queensland, Australia. Activated sludge was enriched for phages using the clinical strains, and four bacteriophages were isolated. The phages were able to cause lysis in a further three identified clinical isolates. Morphology showed that they were all tailed phages (of the order Caudovirales), two belonging to the family Myoviridae and the others assigned to the Podoviridae and Siphoviridae. Their genomes were sequenced to reveal a doubled stranded DNA topology with genome sizes ranging from 42 kb to 65 kb. In isolating and characterising these novel phages, we directed our efforts toward the development and use of these phages as candidates for phage therapy as an alternative strategy for the management or elimination of these pathogenic strains. Here we describe novel phage candidates for potential therapeutic treatment of MDR Australian clinical isolates of P. aeruginosa.

The presence of plasmids in bacterial hosts alters phage isolation and infectivity.

Antibiotic resistance genes are often carried by plasmids, which spread intra- and inter genera bacterial populations, and also play a critical role in bacteria conferring phage resistance. However, it remains unknown about the influence of plasmids present in bacterial hosts on phage isolation and subsequent infectivity. In this study, using both Escherichia coli and Pseudomonas putida bacteria containing different plasmids, eight phages were isolated and characterized in terms of phage morphology and host range analysis, in conjunction with DNA and protein sequencing. We found that plasmids can influence both the phage isolation process and phage infectivity. In particular, the isolated phages exhibited different phage plaquing infectivity towards the same bacterial species containing different plasmids. Furthermore, the presence of plasmids was found to alter the expression of bacteria membrane protein, which correlates with bacterial cell surface receptors recognized by phages, thus affecting phage isolation and infectivity. Given the diverse and ubiquitous nature of plasmids, our findings highlight the need to consider plasmids as factors that can influence both phage isolation and infectivity.

Characterizing the premise plumbing microbiome in both water and biofilms of a 50-year-old building.

The premise plumbing portion of drinking water distribution systems (DWDS) has several characteristics that may favor microbial growth in the form of biofilms. These microbial communities are implicated as infectious sources for the spread of opportunistic waterborne pathogens by supporting their complex ecology and transmission through DWDS outlets to susceptible individuals. However, there is limited understanding of the drinking water biofilms in real premise plumbing networks due to challenges with accessibility. Using a combination of culture-dependent and culture-independent approaches, this study comprehensively characterized the premise plumbing microbiome of a 50-year-old university building, inclusive of water and biofilm samples. Microbial diversity in the water samples were more taxonomically diverse in comparison to the mature drinking water biofilms, which were dominated with biofilm-formers and opportunistic pathogens, such as Mycobacterium spp. A model opportunistic pathogen, Legionella spp., was only detectable in water samples using quantitative PCR but could not be detected in any of the drinking water biofilms using either qPCR or culture-dependent approaches, highlighting the limitations of detection methods in these environments. This study presents preliminary findings on the microbial dynamics and complexity in premise plumbing networks, which may support public health management and the development of strategies to eliminate microbial risks to human health.

Building Better Bacteriophage with Biofoundries to Combat Antibiotic-Resistant Bacteria.

Resistance to antibiotics is an escalating global crisis, presenting a major health, social, and economic burden. An underexplored alternative to antibiotic treatment is phage therapy whereby bacteriophages are used to infect and kill pathogenic multidrug-resistant (MDR) bacteria. A primary challenge is the highly specific infectivity range of phages that can limit their ability to infect across different bacterial strains. Synthetic biology can enable the design, modification, and synthesis of phages with improved antimicrobial performance and efficacy to help realize novel strategies to study and treat bacterial infectious diseases, including those caused by MDR pathogens. In this perspective article, we discuss the potential for an innovative synthetic biology approach to enhance phage therapeutics and the role a biofoundry can play in bringing phage therapy to fruition.

Thermal stress modifies the marine sponge virome.

Marine sponges can form stable partnerships with a wide diversity of microbes and viruses, and this high intraspecies symbiont specificity makes them ideal models for exploring how host-associated viromes respond to changing environmental conditions. Here we exposed the abundant Great Barrier Reef sponge Rhopaloiedes odorabile to elevated seawater temperature for 48 h and utilised a metaviromic approach to assess the response of the associated viral community. An increase in endogenous retro-transcribing viruses within the Caulimorviridae and Retroviridae families was detected within the first 12 h of exposure to 32 °C, and a 30-fold increase in retro-transcribing viruses was evident after 48 h at 32 °C. Thermally stressed sponges also exhibited a complete loss of ssDNA viruses which were prevalent in field samples and sponges from the control temperature treatment. Despite these viromic changes, functional analysis failed to detect any loss or gain of auxiliary metabolic genes, indicating that viral communities are not providing a direct competitive advantage to their host under thermal stress. In contrast, endogenous sponge retro-transcribing viruses appear to be replicating under thermal stress, and consistent with retroviral infections in other organisms, may be contributing to the previously described rapid decline in host health evident at elevated temperature.

Viruses in Marine Ecosystems: From Open Waters to Coral Reefs.

Viruses infect all kingdoms of marine life from bacteria to whales. Viruses in the world's oceans play important roles in the mortality of phytoplankton, and as drivers of evolution and biogeochemical cycling. They shape host population abundance and distribution and can lead to the termination of algal blooms. As discoveries about this huge reservoir of genetic and biological diversity grow, our understanding of the major influences viruses exert in the global marine environment continues to expand. This chapter discusses the key discoveries that have been made to date about marine viruses and the current direction of this field of research.

Reef invertebrate viromics: diversity, host specificity and functional capacity.

Recent metagenomic analyses have revealed a high diversity of viruses in the pelagic ocean and uncovered clear habitat-specific viral distribution patterns. Conversely, similar insights into the composition, host specificity and function of viruses associated with marine organisms have been limited by challenges associated with sampling and computational analysis. Here, we performed targeted viromic analysis of six coral reef invertebrate species and their surrounding seawater to deliver taxonomic and functional profiles of viruses associated with reef organisms. Sponges and corals' host species-specific viral assemblages with low sequence identity to known viral genomes. While core viral genes involved in capsid formation, tail structure and infection mechanisms were observed across all reef samples, auxiliary genes including those involved in herbicide resistance and viral pathogenesis pathways such as host immune suppression were differentially enriched in reef hosts. Utilising a novel OTU based assessment, we also show a prevalence of dsDNA viruses belonging to the Mimiviridae, Caudovirales and Phycodnaviridae in reef environments and further highlight the abundance of ssDNA viruses belonging to the Circoviridae, Parvoviridae, Bidnaviridae and Microviridae in reef invertebrates. These insights into coral reef viruses provide an important framework for future research into how viruses contribute to the health and evolution of reef organisms.

Coral-associated viral communities show high levels of diversity and host auxiliary functions.

Stony corals (Scleractinia) are marine invertebrates that form the foundation and framework upon which tropical reefs are built. The coral animal associates with a diverse microbiome comprised of dinoflagellate algae and other protists, bacteria, archaea, fungi and viruses. Using a metagenomics approach, we analysed the DNA and RNA viral assemblages of seven coral species from the central Great Barrier Reef (GBR), demonstrating that tailed bacteriophages of the Caudovirales dominate across all species examined, and ssDNA viruses, notably the Microviridae, are also prevalent. Most sequences with matches to eukaryotic viruses were assigned to six viral families, including four Nucleocytoplasmic Large DNA Viruses (NCLDVs) families: Iridoviridae, Phycodnaviridae, Mimiviridae, and Poxviridae, as well as Retroviridae and Polydnaviridae. Contrary to previous findings, Herpesvirales were rare in these GBR corals. Sequences of a ssRNA virus with similarities to the dinornavirus, Heterocapsa circularisquama ssRNA virus of the Alvernaviridae that infects free-living dinoflagellates, were observed in three coral species. We also detected viruses previously undescribed from the coral holobiont, including a virus that targets fungi associated with the coral species Acropora tenuis. Functional analysis of the assembled contigs indicated a high prevalence of latency-associated genes in the coral-associated viral assemblages, several host-derived auxiliary metabolic genes (AMGs) for photosynthesis (psbA, psbD genes encoding the photosystem II D1 and D2 proteins respectively), as well as potential nematocyst toxins and antioxidants (genes encoding green fluorescent-like chromoprotein). This study expands the currently limited knowledge on coral-associated viruses by characterising viral composition and function across seven GBR coral species.

A PCR-Based Assay Targeting the Major Capsid Protein Gene of a Dinorna-Like ssRNA Virus That Infects Coral Photosymbionts.

The coral-Symbiodinium association is a critical component of coral reefs as it is the main primary producer and builds the reef's 3-dimensional structure. A breakdown of this endosymbiosis causes a loss of the dinoflagellate photosymbiont, Symbiodinium, and/or its photosynthetic pigments from the coral tissues (i.e., coral bleaching), and can lead to coral mortality. Coral bleaching has mostly been attributed to environmental stressors, and in some cases to bacterial infection. Viral lysis of Symbiodinium has been proposed as another possible cause of some instances of coral bleaching, but this hypothesis has not yet been experimentally confirmed. In this study, we used coral virome data to develop a novel PCR-based assay for examining the presence and diversity of a single-stranded RNA (ssRNA) virus by targeting its major capsid protein (MCP) gene. Illumina sequence analysis of amplicons obtained with novel primers showed 99.8% of the reads had the closest taxonomic affinity with the MCP gene of the virus, Heterocapsa circularisquama RNA virus (HcRNAV) known to infect dinoflagellates, indicating that dinorna-like viruses are commonly associated with corals on the Great Barrier Reef. A phylogenetic analysis of MCP gene sequences revealed strong coral species specificity of viral operational taxon units (OTUs). This assay allows a relatively easy and rapid evaluation of the presence and diversity of this particular viral group and will assist in enhancing our understanding of the role of viral lysis in coral bleaching.

Marine Prasinoviruses and Their Tiny Plankton Hosts: A Review.

Viruses play a crucial role in the marine environment, promoting nutrient recycling and biogeochemical cycling and driving evolutionary processes. Tiny marine phytoplankton called prasinophytes are ubiquitous and significant contributors to global primary production and biomass. A number of viruses (known as prasinoviruses) that infect these important primary producers have been isolated and characterised over the past decade. Here we review the current body of knowledge about prasinoviruses and their interactions with their algal hosts. Several genes, including those encoding for glycosyltransferases, methyltransferases and amino acid synthesis enzymes, which have never been identified in viruses of eukaryotes previously, have been detected in prasinovirus genomes. The host organisms are also intriguing; most recently, an immunity chromosome used by a prasinophyte in response to viral infection was discovered. In light of such recent, novel discoveries, we discuss why the cellular simplicity of prasinophytes makes for appealing model host organism-virus systems to facilitate focused and detailed investigations into the dynamics of marine viruses and their intimate associations with host species. We encourage the adoption of the prasinophyte Ostreococcus and its associated viruses as a model host-virus system for examination of cellular and molecular processes in the marine environment.

Unraveling the microbial processes of black band disease in corals through integrated genomics.

Coral disease outbreaks contribute to the ongoing degradation of reef ecosystems, however, microbial mechanisms underlying the onset and progression of most coral diseases are poorly understood. Black band disease (BBD) manifests as a cyanobacterial-dominated microbial mat that destroys coral tissues as it rapidly spreads over coral colonies. To elucidate BBD pathogenesis, we apply a comparative metagenomic and metatranscriptomic approach to identify taxonomic and functional changes within microbial lesions during in-situ development of BBD from a comparatively benign stage termed cyanobacterial patches. Results suggest that photosynthetic CO2-fixation in Cyanobacteria substantially enhances productivity of organic matter within the lesion during disease development. Photosynthates appear to subsequently promote sulfide-production by Deltaproteobacteria, facilitating the major virulence factor of BBD. Interestingly, our metagenome-enabled transcriptomic analysis reveals that BBD-associated cyanobacteria have a putative mechanism that enables them to adapt to higher levels of hydrogen sulfide within lesions, underpinning the pivotal roles of the dominant cyanobacterium within the polymicrobial lesions during the onset of BBD. The current study presents sequence-based evidence derived from whole microbial communities that unravel the mechanism of development and progression of BBD.

HoloVir: A Workflow for Investigating the Diversity and Function of Viruses in Invertebrate Holobionts.

Abundant bioinformatics resources are available for the study of complex microbial metagenomes, however their utility in viral metagenomics is limited. HoloVir is a robust and flexible data analysis pipeline that provides an optimized and validated workflow for taxonomic and functional characterization of viral metagenomes derived from invertebrate holobionts. Simulated viral metagenomes comprising varying levels of viral diversity and abundance were used to determine the optimal assembly and gene prediction strategy, and multiple sequence assembly methods and gene prediction tools were tested in order to optimize our analysis workflow. HoloVir performs pairwise comparisons of single read and predicted gene datasets against the viral RefSeq database to assign taxonomy and additional comparison to phage-specific and cellular markers is undertaken to support the taxonomic assignments and identify potential cellular contamination. Broad functional classification of the predicted genes is provided by assignment of COG microbial functional category classifications using EggNOG and higher resolution functional analysis is achieved by searching for enrichment of specific Swiss-Prot keywords within the viral metagenome. Application of HoloVir to viral metagenomes from the coral Pocillopora damicornis and the sponge Rhopaloeides odorabile demonstrated that HoloVir provides a valuable tool to characterize holobiont viral communities across species, environments, or experiments.

Genetic, morphological and growth characterisation of a new Roseofilum strain (Oscillatoriales, Cyanobacteria) associated with coral black band disease.

Black band disease (BBD) is a common disease of reef-building corals with a worldwide distribution that causes tissue loss at a rate of up to 3 cm/day. Critical for a mechanistic understanding of the disease's aetiology is the cultivation of its proposed pathogen, filamentous cyanobacteria (genus Roseofilum). Here, we optimise existing protocols for the isolation and cultivation of Roseofilum cyanobacteria using a new strain from the central Great Barrier Reef. We demonstrate that the isolation of this bacterium via inoculation onto agar plates was highly effective with a low percentage agar of 0.6% and that growth monitoring was most sensitive with fluorescence measurements of chlorophyll-a (440/685 nm). Cell growth curves in liquid and solid media were generated for the first time for this cyanobacterium and showed best growth rates for the previously untested L1-medium (growth rate k = 0.214 biomass/day; doubling time t gen = 4.67 days). Our results suggest that the trace metals contained in L1-medium maximise biomass increase over time for this cyanobacterium. Since the newly isolated Roseofilum strain is genetically closest to Pseudoscillatoria coralii, but in terms of pigmentation and cell size closer to Roseofilum reptotaenium, we formally merge the two species into a single taxon by providing an emended species description, Roseofilum reptotaenium (Rasoulouniriana) Casamatta emend. Following this optimized protocol is recommended for fast isolation and cultivation of Roseofilum cyanobacteria, for growth curve generation in strain comparisons and for maximisation of biomass in genetic studies.

CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease.

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called "CRISPRs." Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen.

Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

Metagenomic characterization of viral communities in corals: mining biological signal from methodological noise.

Reef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities and requires specialized methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this minireview, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double- and single-stranded DNA and RNA viruses. The identified viruses are dominated by double-stranded DNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral and other eukaryotes in close association.