A mechanistic framework for complex microbe-host symbioses.

Virtually all multicellular organisms on Earth live in symbiotic associations with complex microbial communities: the microbiome. This ancient relationship is of fundamental importance for both the host and the microbiome. Recently, the analyses of numerous microbiomes have revealed an incredible diversity and complexity of symbionts, with different mechanisms identified as potential drivers of this diversity. However, the interplay of ecological and evolutionary forces generating these complex associations is still poorly understood. Here we explore and summarise the suite of ecological and evolutionary mechanisms identified as relevant to different aspects of microbiome complexity and diversity. We argue that microbiome assembly is a dynamic product of ecology and evolution at various spatio-temporal scales. We propose a theoretical framework to classify mechanisms and build mechanistic host-microbiome models to link them to empirical patterns. We develop a cohesive foundation for the theoretical understanding of the combined effects of ecology and evolution on the assembly of complex symbioses.

Decoupling of strain- and intrastrain-level interactions of microbiomes in a sponge holobiont.

Holobionts are highly organized assemblages of eukaryotic hosts, cellular microbial symbionts, and viruses, whose interactions and evolution involve complex biological processes. It is largely unknown which specific determinants drive similarity or individuality in genetic diversity between holobionts. Here, we combine short- and long-read sequencing and DNA-proximity-linkage technologies to investigate intraspecific diversity of the microbiomes, including host-resolved viruses, in individuals of a model marine sponge. We find strong impacts of the sponge host and the cellular hosts of viruses on strain-level organization of the holobiont, whereas substantial overlap in nucleotide diversity between holobionts suggests frequent exchanges of microbial cells and viruses at intrastrain level in the local sponge population. Immune-evasive arms races likely restricted virus-host co-evolution at the intrastrain level, generated holobiont-specific genome variations, and linked virus-host genetics through recombination. Our work shows that a decoupling of strain- and intrastrain-level interactions is a key factor in the genetic diversification of holobionts.

The Effect of Face Mask Wear on the Ocular Surface and Contact Lens Microbiome.

OBJECTIVES: As face mask wear can result in the redirection of nasal and oral exhalation toward the ocular region, this study investigated the impact of face mask wear on the conjunctiva, eyelid margin, and contact lens (CL) surface microbiome. METHODS: In this prospective, cross-over study, experienced CL wearers (N=20) were randomized to wear a face mask for 6 hr/day (minimum) for a week or no mask for a week. The conjunctiva, eyelid, and CLs were then sampled. After a 1-week washout period, participants were crossed over into the alternate treatment for 1 week and sampling was repeated. Sampling was bilateral and randomly assigned to be processed for culturing or 16S ribosomal(r) RNA gene sequencing. RESULTS: Culturing showed no effect of mask wear on the average number of bacterial colonies isolated on the conjunctiva, eyelid, or CL, but there was increased isolation of Staphylococcus capitis on CL samples with mask wear (P=0.040). Culture-independent sequencing found differences in the taxonomic complexity and bacterial composition between the three sites (P<0.001), but there was no effect of bacterial diversity within and between sites. Mask wear did not impact dry eye or CL discomfort, but increased ocular surface staining was reported (P=0.035). CONCLUSIONS: Mask wear did not substantially alter the microbiome of the conjunctiva, eyelid margin, or CL surfaces in uncompromised healthy eyes.

Marine heatwave-driven mass mortality and microbial community reorganisation in an ecologically important temperate sponge.

Marine heatwaves (MHWs) are increasing in frequency, duration and intensity, disrupting global marine ecosystems. While most reported impacts have been in tropical areas, New Zealand experienced its strongest and longest MHW in 2022, profoundly affecting marine sponges. Sponges are vital to rocky benthic marine communities, with their abundance influencing ecosystem functioning. This study examines the impact of this MHW on the photosynthetic sponge Cymbastella lamellata in Fiordland, New Zealand. We describe the extent, physiological responses, mortality, microbial community changes and ecological impact of this MHW on C. lamellata. The Fiordland MHW reached a maximum temperature of 4.4°C above average, lasting for 259 days. Bleaching occurred in >90% of the C. lamellata Fiordland population. The population size exceeded 66 million from 5 to 25 m, making this the largest bleaching event of its kind ever recorded. We identified the photosynthetic symbiont as a diatom, and bleached sponges had reduced photosynthetic efficiency. Post-MHW surveys in 2023 found that over 50% of sponges at sampling sites had died but that the remaining sponges had mostly recovered from earlier bleaching. Using a simulated MHW experiment, we found that temperature stress was a driver of necrosis rather than bleaching, despite necrosis only rarely being observed in the field (<2% of sponges). This suggests that bleaching may not be the cause of the mortality directly. We also identified a microbial community shift in surviving sponges, which we propose represents a microbial-mediated adaptive response to MHWs. We also found that C. lamellata are key contributors of dissolved organic carbon to the water column, with their loss likely impacting ecosystem function. We demonstrate the potential for MHWs to disrupt key marine phyla in temperate regions, highlighting how susceptible temperate sponges globally might be to MHWs.

Refining microbial community metabolic models derived from metagenomics using reference-based taxonomic profiling.

Characterization of microbial community metabolic output is crucial to understanding their functions. Construction of genome-scale metabolic models from metagenome-assembled genomes (MAG) has enabled prediction of metabolite production by microbial communities, yet little is known about their accuracy. Here, we examined the performance of two approaches for metabolite prediction from metagenomes, one that is MAG-guided and another that is taxonomic reference-guided. We applied both on shotgun metagenomics data from human and environmental samples, and validated findings in the human samples using untargeted metabolomics. We found that in human samples, where taxonomic profiling is optimized and reference genomes are readily available, when number of input taxa was normalized, the reference-guided approach predicted more metabolites than the MAG-guided approach. The two approaches showed significant overlap but each identified metabolites not predicted in the other. Pathway enrichment analyses identified significant differences in inferences derived from data based on the approach, highlighting the need for caution in interpretation. In environmental samples, when the number of input taxa was normalized, the reference-guided approach predicted more metabolites than the MAG-guided approach for total metabolites in both sample types and non-redundant metabolites in seawater samples. Nonetheless, as was observed for the human samples, the approaches overlapped substantially but also predicted metabolites not observed in the other. Our findings report on utility of a complementary input to genome-scale metabolic model construction that is less computationally intensive forgoing MAG assembly and refinement, and that can be applied on shallow shotgun sequencing where MAGs cannot be generated.IMPORTANCELittle is known about the accuracy of genome-scale metabolic models (GEMs) of microbial communities despite their influence on inferring community metabolic outputs and culture conditions. The performance of GEMs for metabolite prediction from metagenomes was assessed by applying two approaches on shotgun metagenomics data from human and environmental samples, and validating findings in the human samples using untargeted metabolomics. The performance of the approach was found to be dependent on sample type, but collectively, the reference-guided approach predicted more metabolites than the MAG-guided approach. Despite the differences, the predictions from the approaches overlapped substantially but each identified metabolites not predicted in the other. We found significant differences in biological inferences based on the approach, with some examples of uniquely enriched pathways in one group being invalidated when using the alternative approach, highlighting the need for caution in interpretation of GEMs.

Seaweeds as holobionts: Current state, challenges, and potential applications.

Seaweeds play a strong ecological and economical role along the world's coastlines, where they support industries (e.g., aquaculture, bioproducts) and essential ecosystem services (e.g., biodiversity, fisheries, carbon capture). Evidence from wild and cultured seaweeds suggests that microorganisms play crucial roles in their health and functioning, prompting the need for considering seaweeds and their microbiome as a coherent entity or "holobiont." Here we show that the number of studies investigating seaweed hosts and their microbiome have increased in the last two decades. This likely reflects the increase in the appreciation of the importance of microbiomes for eukaryotic hosts, improved molecular approaches used to characterize their interactions, and increasing interest in commercial use of seaweeds. However, although increasing, most studies of seaweed holobionts have focused on (i) a few seaweed species of ecological or commercial significance, (ii) interactions involving only bacteria, and (iii) descriptive rather than experimental approaches. The relatively few experimental studies have mostly focused on manipulating abiotic factors to examine responses of seaweeds and their microbiome. Of the few studies that directly manipulated microorganisms to investigate their effects on seaweeds, most were done in laboratory or aquaria. We emphasize the need to move beyond the descriptions of patterns to experimental approaches for understanding causation and mechanisms. We argue that such experimental approaches are necessary for a better understanding of seaweed holobionts, for management actions for wild and cultivated seaweeds, and to better integrate studies of seaweed holobionts with the broader fields of seaweed ecology and biology, which are strongly experimental.

Non-selective microbiota reduction after the elicitation of a seaweed's immune response.

Pattern-triggered immunity (PTI) is an integral part of the innate immune system of many eukaryotic hosts, assisting in the defence against pathogen invasions. In plants and animals, PTI exerts a selective pressure on the microbiota that can alter community composition. However, the effect of PTI on the microbiota for non-model hosts, including seaweeds, remains unknown. Using quantitative polymerase chain reaction complemented with 16S rRNA gene and transcript amplicon sequencing, this study profiled the impact that PTI of the red seaweed Gracilaria gracilis has on its microbiota. PTI elicitation with agar oligosaccharides resulted in a significant reduction in the number of bacteria (by >75% within 72 h after treatment). However, the PTI elicitation did not cause any significant difference in the community diversity or structure. These findings demonstrated that PTI can be non-selective, and this might help to maintain a stable microbiota by uniformly reducing bacterial loads.

Bacteria associated with the in hospite Symbiodiniaceae's phycosphere.

Symbiotic interactions between Symbiodiniaceae and bacteria are still poorly explored, especially those in hospite. Here, we adapted a technique that allows for the enrichment of intact and metabolically active in hospite Symbiodiniaceae cells (ihSC) and their associated bacteria from the tissue of the model coral Pocillopora damicornis, using a discontinuous gradient of solution of isotonic Percoll (SIP). The ihSC were concentrated in the 50% SIP fraction, as determined by microscopy. The presence of bacteria associated with ihSC was confirmed by fluorescence in situ hybridization, while microbiome analysis indicated that bacteria of the families Halieaceae, Flavobacteriaceae, and Alcanivoraceae are significantly associated with ihSC. Extracellular vesicles that could be exuding molecules were detected on the symbiosome membranes. Our technique and data contribute to elucidate ihSC-bacteria interactions.

Genomic characterization of extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli from urban wastewater in Australia.

This study investigates extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli isolates from Sydney's wastewater. These isolates exhibit resistance to critical antibiotics and harbor novel resistance mechanisms. The findings highlight the importance of wastewater-based surveillance in monitoring resistance beyond the clinical setting.

Functional guilds and drivers of diversity in seaweed-associated bacteria.

Comparisons of functional and taxonomic profiles from bacterial communities in different habitats have suggested the existence of functional guilds composed of taxonomically or phylogenetically distinct members. Such guild membership is, however, rarely defined and the factors that drive functional diversity in bacteria remain poorly understood. We used seaweed-associated bacteria as a model to shed light on these important aspects of community ecology. Using a large dataset of over 1300 metagenome-assembled genomes from 13 seaweed species we found substantial overlap in the functionality of bacteria coming from distinct taxa, thus supporting the existence of functional guilds. This functional equivalence between different taxa was particularly pronounced when only functions involved in carbohydrate degradation were considered. We further found that bacterial taxonomy is the dominant driver of functional differences between bacteria and that seaweed species or seaweed type (i.e. brown, red and green) had relatively stronger impacts on genome functionality for carbohydrate-degradation functions when compared to all other cellular functions. This study provides new insight into the factors underpinning the functional diversity of bacteria and contributes to our understanding how community function is generated from individual members.

Effect of marine heatwaves and warming on kelp microbiota influence trophic interactions.

The range-expansion of tropical herbivores due to ocean warming can profoundly alter temperate reef communities by overgrazing the seaweed forests that underpin them. Such ecological interactions may be mediated by changes to seaweed-associated microbiota in response to warming, but empirical evidence demonstrating this is rare. We experimentally simulated ocean warming and marine heatwaves (MHWs) to quantify effects on two dominant temperate seaweed species and their microbiota, as well as grazing by a tropical herbivore. The kelp Ecklonia radiata's microbiota in sustained warming and MHW treatments was enriched with microorganisms associated with seaweed disease and tissue degradation. In contrast, the fucoid Sargassum linearifolium's microbiota was unaffected by temperature. Consumption by the tropical sea-urchin Tripneustes gratilla was greater on Ecklonia where the microbiota had been altered by higher temperatures, while Sargassum's consumption was unaffected. Elemental traits (carbon, nitrogen), chemical defences (phenolics) and tissue bleaching of both seaweeds were generally unaffected by temperature. Effects of warming and MHWs on seaweed holobionts (host plus its microbiota) are likely species-specific. The effect of increased temperature on Ecklonia's microbiota and subsequent increased consumption suggest that changes to kelp microbiota may underpin kelp-herbivore interactions, providing novel insights into potential mechanisms driving change in species' interactions in warming oceans.

The impact of interspecific competition on the genomic evolution of Phaeobacter inhibens and Pseudoalteromonas tunicata during biofilm growth.

Interspecific interactions in biofilms have been shown to cause the emergence of community-level properties. To understand the impact of interspecific competition on evolution, we deep-sequenced the dispersal population of mono- and co-culture biofilms of two antagonistic marine bacteria (Phaeobacter inhibens 2.10 and Pseudoalteromononas tunicata D2). Enhanced phenotypic and genomic diversification was observed in the P. tunicata D2 populations under both mono- and co-culture biofilms in comparison to P. inhibens 2.10. The genetic variation was exclusively due to single nucleotide variants and small deletions, and showed high variability between replicates, indicating their random emergence. Interspecific competition exerted an apparent strong positive selection on a subset of P. inhibens 2.10 genes (e.g., luxR, cobC, argH, and sinR) that could facilitate competition, while the P. tunicata D2 population was genetically constrained under competition conditions. In the absence of interspecific competition, the P. tunicata D2 replicate populations displayed high levels of mutations affecting the same genes involved in cell motility and biofilm formation. Our results show that interspecific biofilm competition has a complex impact on genomic diversification, which likely depends on the nature of the competing strains and their ability to generate genetic variants due to their genomic constraints.

Not all parents are the same: Diverse strategies of symbiont transmission in seaweeds.

Different marine seaweed species have been shown to harbour specific bacterial communities, however, the extent to which vertical symbiont transmission from parents to offspring contributes to host-specificity is unclear. Here we use fluorescence and electron microscopy as well as 16S rRNA gene-based community analysis to investigate symbiont transmission in members of the three major seaweed groups (green Chlorophyta, red Rhodophyta and brown Phaeophyceae). We found seaweeds employ diverse strategies to transfer symbionts to their progeny. For instance, the green Ulva australis does not appear to have the capacity for vertical transmission. In contrast, the brown Phyllospora comosa adopts a non-selective vertical transmission. The red Delisea pulchra demonstrates weak selectivity in symbiont transmission, while the brown Hormosira banksii exhibits a strongly selective symbiont transfer. Mucilage on the gametes appears to facilitate vertical transmission and transferred bacteria have predicted properties that could support early development of the seaweeds. Previous meta-analysis has indicated that vertical transmission is rare in aquatic compared to terrestrial environments, however, our results contribute to the growing evidence that this might not be the case and that instead vertical transmission with various degrees of symbiont selection occurs in the ecologically important group of seaweeds.

Ocular microbiome changes in dry eye disease and meibomian gland dysfunction.

The most common and chronic ocular problem of aging is dry eye disease (DED) and the associated condition of meibomian gland dysfunction (MGD). The resident ocular surface bacteria may have a role in maintaining homeostasis and perturbation may contribute to disease development. The aim of this study was to compare the microbiomes of the conjunctiva and eyelid margin in humans with mild and moderate DED and controls using 16 S rRNA gene sequencing. The conjunctiva and lid margin of three cohorts (N = 60; MGD, MGD with lacrimal dysfunction [MGD + LD] and controls) were swabbed bilaterally three times over three months. Microbial communities were analysed by extracting DNA and sequencing the V3-V4 region of the 16 S ribosomal RNA gene using the Illumina MiSeq platform. Sequences were quality filtered, clustered into amplicon sequence variants (ASVs) using UNOISE algorithm and taxonomically classified using a Bayesian Last Common Ancestor (BCLA) algorithm against the GTDB 2207 database. The overall microbial communities of the MGD, MGD + LD and control groups were significantly different from each other (P = 0.001). The MGD and MGD + LD dry eye groups showed greater variability between individuals compared to the control (PERMDISP, P < 0.01). There was decreased richness and diversity in females compared to males for the conjunctiva (P < 0.04) and eyelid margin (P < 0.018). The conjunctiva in the MGD + LD group had more abundant Pseudomonas azotoformans, P. oleovorans and Caballeronia zhejiangensis compared to MGD and control (P < 0.05), while the MGD group had more abundant Corynebacterium macginleyi and C. kroppenstedtii compared to control (P < 0.05). The lid margin in MGD was more abundant in C. macginleyi, C. accolens, and C. simulans compared to the MGD + LD and control (P < 0.05). There were differences in the overall microbial community composition and certain taxa, including increased levels of lipophilic bacteria, on the conjunctiva and eyelid margin in mild to moderate DED/MGD compared to controls. DED/MGD was also associated with a reduced bacterial richness and diversity in females.

The microbiome of the sponge Aplysina caissara in two sites with different levels of anthropogenic impact.

Despite the important roles that marine sponges play in ecosystem functioning and structuring, little is known about how the sponge holobiont responds to local anthropogenic impacts. Here we assess the influence of an impacted environment (Praia Preta) on the microbial community associated with the endemic sponge Aplysina caissara in comparison to a less-impacted area (Praia do Guaecá) from the coast of São Paulo state (Brazil, southwestern Atlantic coast). We hypothesized that the local anthropogenic impacts will change the microbiome of A. caissara and that the community assembly will be driven by a different process (i.e. deterministic versus stochastic) under distinct levels of impact. The microbiome at the amplicon sequence variants level was found to be statistically distinct between sponges from the different sites, and this was also seen for the microbial communities of the surrounding seawater and sediments. Microbial communities of A. caissara from both sites were found to be assembled by deterministic processes, even though the sites presented distinct anthropogenic impacts, showing a pivotal role of the sponge host in selecting its own microbiome. Overall, this study revealed that local anthropogenic impacts altered the microbiome of A. caissara; however, assembly processes are largely determined by the sponge host.

Marine heatwave conditions drive carryover effects in a temperate sponge microbiome and developmental performance.

Marine heatwaves are increasingly subjecting organisms to unprecedented stressful conditions, but the biological consequences of these events are still poorly understood. Here we experimentally tested the presence of carryover effects of heatwave conditions on the larval microbiome, settlers growth rate and metamorphosis duration of the temperate sponge Crella incrustans. The microbial community of adult sponges changed significantly after ten days at 21°C. There was a relative decrease in symbiotic bacteria, and an increase in stress-associated bacteria. Sponge larvae derived from control sponges were mainly characterised by a few bacterial taxa also abundant in adults, confirming the occurrence of vertical transmission. The microbial community of sponge larvae derived from heatwave-exposed sponges showed significant increase in the endosymbiotic bacteria Rubritalea marina. Settlers derived from heatwave-exposed sponges had a greater growth rate under prolonged heatwave conditions (20 days at 21°C) compared to settlers derived from control sponges exposed to the same conditions. Moreover, settler metamorphosis was significantly delayed at 21°C. These results show, for the first time, the occurrence of heatwave-induced carryover effects across life-stages in sponges and highlight the potential role of selective vertical transmission of microbes in sponge resilience to extreme thermal events.

Future ocean conditions induce necrosis, microbial dysbiosis and nutrient cycling imbalance in the reef sponge Stylissa flabelliformis.

Oceans are rapidly warming and acidifying in the context of climate change, threatening sensitive marine biota including coral reef sponges. Ocean warming (OW) and ocean acidification (OA) can impact host health and associated microbiome, but few studies have investigated these effects, which are generally studied in isolation, on a specific component of the holobiont. Here we present a comprehensive view of the consequences of simultaneous OW and OA for the tropical sponge Stylissa flabelliformis. We found no interactive effect on the host health or microbiome. Furthermore, OA (pH 7.6 versus pH 8.0) had no impact, while OW (31.5 °C versus 28.5 °C) caused tissue necrosis, as well as dysbiosis and shifts in microbial functions in healthy tissue of necrotic sponges. Major taxonomic shifts included a complete loss of archaea, reduced proportions of Gammaproteobacteria and elevated relative abundances of Alphaproteobacteria. OW weakened sponge-microbe interactions, with a reduced capacity for nutrient exchange and phagocytosis evasion, indicating lower representations of stable symbionts. The potential for microbially-driven nitrogen and sulphur cycling was reduced, as was amino acid metabolism. Crucially, the dysbiosis annihilated the potential for ammonia detoxification, possibly leading to accumulation of toxic ammonia, nutrient imbalance, and host tissue necrosis. Putative defence against reactive oxygen species was greater at 31.5 °C, perhaps as microorganisms capable of resisting temperature-driven oxidative stress were favoured. We conclude that healthy symbiosis in S. flabelliformis is unlikely to be disrupted by future OA but will be deeply impacted by temperatures predicted for 2100 under a "business-as-usual" carbon emission scenario.

Identification, classification, and functional characterization of novel sponge-associated acidimicrobiial species.

Sponges are known to harbour an exceptional diversity of uncultured microorganisms, including members of the phylum Actinobacteriota. While members of the actinobacteriotal class Actinomycetia have been studied intensively due to their potential for secondary metabolite production, the sister class of Acidimicrobiia is often more abundant in sponges. However, the taxonomy, functions, and ecological roles of sponge-associated Acidimicrobiia are largely unknown. Here, we reconstructed and characterized 22 metagenome-assembled genomes (MAGs) of Acidimicrobiia from three sponge species. These MAGs represented six novel species, belonging to five genera, four families, and two orders, which are all uncharacterized (except the order Acidimicrobiales) and for which we propose nomenclature. These six uncultured species have either only been found in sponges and/or corals and have varying degrees of specificity to their host species. Functional gene profiling indicated that these six species shared a similar potential to non-symbiotic Acidimicrobiia with respect to amino acid biosynthesis and utilization of sulfur compounds. However, sponge-associated Acidimicrobiia differed from their non-symbiotic counterparts by relying predominantly on organic rather than inorganic sources of energy, and their predicted capacity to synthesise bioactive compounds or their precursors implicated in host defence. Additionally, the species possess the genetic capacity to degrade aromatic compounds that are frequently found in sponges. The novel Acidimicrobiia may also potentially mediate host development by modulating Hedgehog signalling and by the production of serotonin, which can affect host body contractions and digestion. These results highlight unique genomic and metabolic features of six new acidimicrobiial species that potentially support a sponge-associated lifestyle.

Species-specific relationships between deep sea sponges and their symbiotic Nitrosopumilaceae.

Sponges thrive in the deep, dark and nutrient-depleted ocean and may rely on microbial symbionts for carbon acquisition and energy generation. However, these symbiotic relationships remain largely unexplored. In this study, we analyze the microbiome of deep-sea sponges and show that ammonia-oxidizing archaea (AOA) of the family Nitrosopumilaceae make up at least 75% of the microbial communities of the sponges Aphrocallistes sp., Farrea sp. and Paratimea sp.. Given the known autotrophic metabolism of AOAs, this implies that these sponge holobionts can have the capacity for primary production in the deep-sea. We also show that specific AOA lineages are highly specific towards their hosts, hinting towards an unprecedent vertical transmission of these symbionts in deep-sea sponges. Our results show that the ecology and evolution of symbiotic relationships in deep-sea sponge is distinct from that of their shallow-water counterparts.

Wastewater-based monitoring reveals geospatial-temporal trends for antibiotic-resistant pathogens in a large urban community.

Antimicrobial resistance (AMR) is one of the top ten global health threats, and current surveillance programs rarely monitor it outside healthcare settings. This limits our ability to understand and manage the spread of AMR. Wastewater testing has the potential to simply, reliably and continuously survey trends in AMR outside the healthcare settings, as it captures biological material from the entire community. To establish and evaluate such a surveillance, we monitored wastewater for four clinically significant pathogens across the urban area of Greater Sydney, Australia. Untreated wastewater from 25 wastewater treatment plants (WWTPs) covering distinct catchment regions of 5.2 million residents was sampled between 2017 and 2019. Isolates for extended-spectrum ß-lactamases-producing Enterobacteriaceae (ESBL-E) were consistently detected, suggesting its endemicity in the community. Isolates for carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) were only occasionally detected. The flow normalized relative (FNR) ESBL-E load was positively correlated with the proportion of the population between 19 and 50 years of age, completion of vocational education and the average length of hospital stay. Collectively, these variables explained only a third of the variance of the FNR ESBL-E load, indicating further, yet-unidentified factors as a contributor to the distribution. About half of the variation in the FNR CRE load was explained by the average length of hospital stay, showing healthcare-related drivers. Interestingly, variation in the FNR VRE load was not correlated to healthcare-related parameters but to the number of schools per 10,000 population. Our study provides insight into how routine wastewater surveillance can be used to understand the factors driving the distribution of AMR in an urban community. Such information can help to manage and mitigate the emergence and spread of AMR in important human pathogens.