Mitigating the impact of the COVID-19 pandemic on Inuit living in Manitoba: community responses.

We document community responses to the COVID-19 pandemic among Inuit living in the province of Manitoba, Canada. This study was conducted by the Manitoba Inuit Association and a Council of Inuit Elders, in partnership with researchers from the University of Manitoba. We present findings from 12 health services providers and decision-makers, collected in 2021.Although Public Health orders led to the closure of the Manitoba Inuit Association's doors to community events and drop-in activities, it also created opportunities for the creation of programming and events delivered virtually and through outreach. The pandemic exacerbated pre-existing health and social system's shortcomings (limited access to safe housing, food insecurity) and trauma-related tensions within the community. The Manitoba Inuit Association achieved unprecedented visibility with the provincial government, receiving bi-weekly reports of COVID-19 testing, results and vaccination rates for Inuit. We conclude that after over a decade of advocacy received with at best tepid enthusiasm by federal and provincial governments, the Manitoba Inuit Association was able effectively advocate for Inuit-centric programming, and respond to Inuit community's needs, bringing visibility to a community that had until then been largely invisible. Still, many programs have been fueled with COVID-19 funding, raising the issue of sustainability.

Cross-jurisdictional pandemic management: providers speaking on the experience of Nunavut Inuit accessing services in Manitoba during the COVID-19 pandemic.

Across Canada, the COVID-19 pandemic placed considerable stress on territorial and provincial healthcare systems. For Nunavut, the need to continue to provide access to critical care to its citizens meant that medical travel to provincial points of care (Edmonton, Winnipeg and Ottawa) had to continue through the pandemic. This complexity created challenges related to the need to keep Nunavut residents safe while accessing care, and to manage the risk of outbreaks in Nunavut resultant from patients returning home. A number of strategies were adopted to mitigate risk, including the expansion of virtual care, self-isolation requirements before returning from Winnipeg, and a level of cross-jurisdictional coordination previously unprecedented. Structural limitations in Nunavut however limited opportunities to expand virtual care, and to allow providers from Manitoba to access the Nunavut's electronic medical records of patients requiring follow up. Thus, known and long-standing issues exacerbated vulnerabilities within the Nunavut healthcare system. We conclude that addressing cross-jurisdictional issues would be well served by the development of a more formal Nunavut-Manitoba agreement (with similar agreements with Ontario and Alberta), outlining mutual obligations and accountabilities.

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesn't degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecule mediated BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis spp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and their role in complex communities should continue to be a priority.

Longitudinal, Multi-Platform Metagenomics Yields a High-Quality Genomic Catalog and Guides an In Vitro Model for Cheese Communities.

Microbiomes are intricately intertwined with human health, geochemical cycles, and food production. While many microbiomes of interest are highly complex and experimentally intractable, cheese rind microbiomes have proven to be powerful model systems for the study of microbial interactions. To provide a more comprehensive view of the genomic potential and temporal dynamics of cheese rind communities, we combined longitudinal, multi-platform metagenomics of three ripening washed-rind cheeses with whole-genome sequencing of community isolates. Sequencing-based approaches revealed a highly reproducible microbial succession in each cheese and the coexistence of closely related Psychrobacter species and enabled the prediction of plasmid and phage diversity and their host associations. In combination with culture-based approaches, we established a genomic catalog and a paired 16-member in vitro washed-rind cheese system. The combination of multi-platform metagenomic time-series data and an in vitro model provides a rich resource for further investigation of cheese rind microbiomes both computationally and experimentally. IMPORTANCE Metagenome sequencing can provide great insights into microbiome composition and function and help researchers develop testable hypotheses. Model microbiomes, such as those composed of cheese rind bacteria and fungi, allow the testing of these hypotheses in a controlled manner. Here, we first generated an extensive longitudinal metagenomic data set. This data set reveals successional dynamics, yields a phyla-spanning bacterial genomic catalog, associates mobile genetic elements with their hosts, and provides insights into functional enrichment of Psychrobacter in the cheese environment. Next, we show that members of the washed-rind cheese microbiome lend themselves to in vitro community reconstruction. This paired metagenomic data and in vitro system can thus be used as a platform for generating and testing hypotheses related to the dynamics within, and the functions associated with, cheese rind microbiomes.

Higher-order interactions shape microbial interactions as microbial community complexity increases.

Non-pairwise interactions, or higher-order interactions (HOIs), in microbial communities have been described as significant drivers of emergent features in microbiomes. Yet, the re-organization of microbial interactions between pairwise cultures and larger communities remains largely unexplored from a molecular perspective but is central to our understanding and further manipulation of microbial communities. Here, we used a bottom-up approach to investigate microbial interaction mechanisms from pairwise cultures up to 4-species communities from a simple microbiome (Hafnia alvei, Geotrichum candidum, Pencillium camemberti and Escherichia coli). Specifically, we characterized the interaction landscape for each species combination involving E. coli by identifying E. coli's interaction-associated mutants using an RB-TnSeq-based interaction assay. We observed a deep reorganization of the interaction-associated mutants, with very few 2-species interactions conserved all the way up to a 4-species community and the emergence of multiple HOIs. We further used a quantitative genetics strategy to decipher how 2-species interactions were quantitatively conserved in higher community compositions. Epistasis-based analysis revealed that, of the interactions that are conserved at all levels of complexity, 82% follow an additive pattern. Altogether, we demonstrate the complex architecture of microbial interactions even within a simple microbiome, and provide a mechanistic and molecular explanation of HOIs.

Kivalliq Inuit women travelling to Manitoba for birthing: findings from the Qanuinngitsiarutiksait study.

BACKGROUND: The Qanuinngitsiarutiksait study aimed to develop detailed profiles of Inuit health service utilization in Manitoba, by Inuit living in Manitoba (approximately 1,500) and by Inuit from the Kivalliq region of Nunavut who travel to Manitoba to access care not available in Nunavut (approximately 16,000 per year). METHODS: We used health administrative data routinely collected in Manitoba for all services provided and developed an algorithm to identify Inuit in the dataset. This paper focused on health services used by Inuit from the Kivalliq for prenatal care and birthing. RESULTS: Our study found that approximately 80 percent of births to women from the Kivalliq region occur in Manitoba, primarily in Winnipeg. When perinatal care and birthing are combined, they constitute one third of all consults happening by Kivalliq residents in Manitoba. For scale, hospitalizations for childbirths to Kivalliq women about to only 5 percent of all childbirth-related hospitalizations in Manitoba. CONCLUSIONS: The practice of evacuating women from the Kivalliq for perinatal care and birthing is rooted in colonialism, rationalized as ensuring that women whose pregnancy is at high risk have access to specialized care not available in Nunavut. While defendable, this practice is costly, and does not provide Inuit women a choice as to where to birth. Attempts at relocating birthing to the north have proven complex to operationalize. Given this, there is an urgent need to develop Inuit-centric and culturally appropriate perinatal and birthing care in Manitoba.

Trends in Inuit health services utilisation in Manitoba: findings from the Qanuinngitsiarutiksait study.

There is a notable lack of research related to trends in Inuit accessing health services throughout the land known as Canada. Given Nunavut's reliance on specialised services provided in the Northwest Territories, Alberta, Manitoba, and Ontario, this gap is particularly problematic, making it more difficult for Nunangat to proactively plan new programs for emerging needs, and for provinces to respond to those needs. The Qanuinngitsiarutiksait study aimed to address this gap by developing detailed profiles of Inuit accessing health services in Manitoba. We used administrative data routinely collected by Manitoba agencies, to support the development of Inuit-centric services. It was conducted in partnership with the Manitoba Inuit Association, and Inuit Elders from Nunavut Canada and Manitoba. We focused on two interrelated cohorts: Kivallirmiut (Inuit from the Kivalliq region of Nunavut) who come to Winnipeg to access specialised services; and Manitobamiut (Inuit already living in Manitoba). Findings show that health services are primarily accessed in Winnipeg. Half of health services accessed by Kivallirmiut are for in-patient care at facilities with the Winnipeg Regional Health Authority. The other half are for advanced out-patient care including specialist consults. For Kivallirmiut, hospitalisation for pregnancy and birth are the most prevalent reasons for hospitalisation, followed by diseases of the respiratory system. Noteworthy, rates of hospitalisation for conditions treatable in primary healthcare for Kivallirmiut are considerably lower than those for Manitobans living in the northern part of the province (where comparable constraints exist). For Inuit adults, rates of hospitalisation for these conditions are comparable to those of Manitobans living in small communities. Inuit living in Manitoba are most often hospitalised for mental health reasons, although other reasons are nearly as prevalent. Our results support the need for more Inuit-centric health programming in Winnipeg.

Unforeseen benefits: outcomes of the Qanuinngitsiarutiksait study.

Scientific publications predominantly focus on research outcomes. Increasingly, community partnerships and relationships are mentioned, especially in research conducted with Indigenous communities. In partnership-based research, Indigenous communities expect researchers to contribute in a multitude of ways that go beyond doing research. This article reports on a series of unforeseen, yet positive contributions realised in the Qanuinngitsiarutiksait study, undertaken between 2015 and 2021. These contributions are different from the main outcomes of the study. Salient unforeseen benefits included the strengthening of the Manitoba Inuit community through hosting community feasts, games, and virtual events; creating opportunities to increase the visibility of Inuit Elders at University public events; supporting the growth of the Manitoba Inuit Association in terms of staff, programmes, and presence at provincial policy tables; leveraging relationships towards the development of Inuit-centric primary healthcare services in Winnipeg; creating a method to identify Inuit in provincial administrative datasets which were used to track COVID-19 infection rates and ensure equity in access to testing and vaccines. As a result, the Manitoba Inuit Association's visibility has increased, and Inuit Elders have become essential contributors of Indigenous knowledge at Manitoba-based events, as First Nations and Metis have been for decades. This transformation appears to be sustainable.

Putting microbial interactions back into community contexts.

Microbial interactions are key aspects of the biology of microbiomes. Recently, there has been a shift in the field towards studying interactions in more representative contexts, whether using multispecies model microbial communities or by looking at interactions in situ. Across diverse microbial systems, these studies have begun to identify common interaction mechanisms. These mechanisms include interactions related to toxic molecules, nutrient competition and cross-feeding, access to metals, signaling pathways, pH changes, and interactions within biofilms. Leveraging technological innovations, many of these studies have used an interdisciplinary approach combining genetic, metabolomic, imaging, and/or microfluidic techniques to gain insight into mechanisms of microbial interactions and into the impact of these interactions on microbiomes.

Native mass spectrometry-based metabolomics identifies metal-binding compounds.

Although metals are essential for the molecular machineries of life, systematic methods for discovering metal-small molecule complexes from biological samples are limited. Here, we describe a two-step native electrospray ionization-mass spectrometry method, in which post-column pH adjustment and metal infusion are combined with ion identity molecular networking, a rule-based data analysis workflow. This method enabled the identification of metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion species with the same chromatographic profiles. As this native electrospray metabolomics approach is suited to the use of any liquid chromatography-mass spectrometry system to explore the binding of any metal, this method has the potential to become an essential strategy for elucidating metal-binding molecules in biology.

Auto-deconvolution and molecular networking of gas chromatography-mass spectrometry data.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.

Bacterial-fungal interactions revealed by genome-wide analysis of bacterial mutant fitness.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Experimental approaches to tracking mobile genetic elements in microbial communities.

Horizontal gene transfer is an important mechanism of microbial evolution and is often driven by the movement of mobile genetic elements between cells. Due to the fact that microbes live within communities, various mechanisms of horizontal gene transfer and types of mobile elements can co-occur. However, the ways in which horizontal gene transfer impacts and is impacted by communities containing diverse mobile elements has been challenging to address. Thus, the field would benefit from incorporating community-level information and novel approaches alongside existing methods. Emerging technologies for tracking mobile elements and assigning them to host organisms provide promise for understanding the web of potential DNA transfers in diverse microbial communities more comprehensively. Compared to existing experimental approaches, chromosome conformation capture and methylome analyses have the potential to simultaneously study various types of mobile elements and their associated hosts. We also briefly discuss how fermented food microbiomes, given their experimental tractability and moderate species complexity, make ideal models to which to apply the techniques discussed herein and how they can be used to address outstanding questions in the field of horizontal gene transfer in microbial communities.

A review of health and wellness studies involving Inuit of Manitoba and Nunavut.

The purpose of this review is to summarise past Inuit health and wellness studies in Manitoba and the Kivalliq region of Nunavut to provide a snapshot of the types of studies available and identify the gaps in knowledge. Research to date has largely been disease-based and often provides comparisons between Indigenous and non-Indigenous people. Distinct Inuit experiences are rarely written about from an Inuit perspective. However, Inuit Tapiriit Kanatami, the national organisation of Inuit in Canada, and Pauktuutit Inuit Women of Canada have been leaders in strengths-based community research and publications that address priorities determined by the Inuit, including the 2018 Inuit Tapiriit Kanatami document National Inuit Strategy on Research (132).

BLANKA: an Algorithm for Blank Subtraction in Mass Spectrometry of Complex Biological Samples.

Multispecies microbiome systems are known to be closely linked to human, animal, and plant life processes. The growing field of metabolomics presents the opportunity to detect changes in overall metabolomic profiles of microbial species interactions. These metabolomic changes provide insight into function of metabolites as they correlate to different species presence and the observed phenotypic changes, but detection of subtle changes is often difficult in samples with complex backgrounds. Natural environments such as soil and food contain many molecules that convolute mass spectrometry-based analyses, and identification of microbial metabolites amongst environmental metabolites is an informatics problem we begin to address here. Our microbes are grown on solid or liquid cheese curd media. This medium, which is necessary for microbial growth, contains high amounts of salts, lipids, and casein breakdown products which make statistical analyses using LC-MS/MS data difficult due to the high background from the media. We have developed a simple algorithm to carry out background subtraction from microbes grown on solid or liquid cheese curd media to aid in our ability to conduct statistical analyses so that we may prioritize metabolites for further structure elucidation. Graphical Abstract .

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.