Increased iron utilization and oxidative stress tolerance in a Vibrio cholerae flrA mutant confers resistance to amoeba predation.

IMPORTANCE: Persistence of V. cholerae in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba A. castellanii. This study explores how a mutant of V. cholerae adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that V. cholerae can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.

Associational Resistance to Predation by Protists in a Mixed Species Biofilm.

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

Environmental Reservoirs of Pathogenic Vibrio spp. and Their Role in Disease: The List Keeps Expanding.

Vibrio species are natural inhabitants of aquatic environments and have complex interactions with the environment that drive the evolution of traits contributing to their survival. These traits may also contribute to their ability to invade or colonize animal and human hosts. In this review, we attempt to summarize the relationships of Vibrio spp. with other organisms in the aquatic environment and discuss how these interactions could potentially impact colonization of animal and human hosts.

Adaptation to an Amoeba Host Leads to Pseudomonas aeruginosa Isolates with Attenuated Virulence.

The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies.

Loss of the Acetate Switch in Vibrio vulnificus Enhances Predation Defense against Tetrahymena pyriformis.

Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with antipredation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defense mechanisms. To identify antipredator strategies, 13 V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan Tetrahymena pyriformis, and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which are toxic to T. pyriformis. As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator arcA was upregulated when iron was available. An ΔarcA deletion mutant of ENV1 accumulated less acetate and, importantly, was sensitive to grazing by T. pyriformis. Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. IMPORTANCE Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve antipredator mechanisms ranging from changing morphology, biofilm formation, and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defense strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is known about the defense mechanisms V. vulnificus expresses against predation. Here, we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism, enabling the production of excess organic acid that is toxic to the protozoan predator T. pyriformis. This is a previously unknown mechanism of predation defense that protects against protozoan predators.

Differential Response of the Microbiome of Pocillopora acuta to Reciprocal Transplantation Within Singapore.

As corals continue to decline globally, particularly due to climate change, it is vital to understand the extent to which their microbiome may confer an adaptive resilience against environmental stress. Corals that survive on the urban reefs of Singapore are ideal candidates to study the association of scleractinians with their microbiome, which in turn can inform reef conservation and management. In this study, we monitored differences in the microbiome of Pocillopora acuta colonies reciprocally transplanted between two reefs, Raffles and Kusu, within the Port of Singapore, where corals face intense anthropogenic impacts. Pocillopora acuta had previously been shown to host distinct microbial communities between these two reefs. Amplicon sequencing (16S rRNA) was used to assess the coral microbiomes at 1, 2, 4, and 10 days post-transplantation. Coral microbiomes responded rapidly to transplantation, becoming similar to those of the local corals at the destination reef within one day at Raffles and within two days at Kusu. Elevated nitrate concentrations were detected at Raffles for the duration of the study, potentially influencing the microbiome's response to transplantation. The persistence of corals within the port of Singapore highlights the ability of corals to adapt to stressful environments. Further, coral resilience appears to coincide with a dynamic microbiome which can undergo shifts in composition without succumbing to dysbiosis.

Microbial predation accelerates granulation and modulates microbial community composition.

BACKGROUND: Bacterial communities are responsible for biological nutrient removal and flocculation in engineered systems such as activated floccular sludge. Predators such as bacteriophage and protozoa exert significant predation pressure and cause bacterial mortality within these communities. However, the roles of bacteriophage and protozoan predation in impacting granulation process remain limited. Recent studies hypothesised that protozoa, particularly sessile ciliates, could have an important role in granulation as these ciliates were often observed in high abundance on surfaces of granules. Bacteriophages were hypothesized to contribute to granular stability through bacteriophage-mediated extracellular DNA release by lysing bacterial cells. This current study investigated the bacteriophage and protozoan communities throughout the granulation process. In addition, the importance of protozoan predation during granulation was also determined through chemical killing of protozoa in the floccular sludge. RESULTS: Four independent bioreactors seeded with activated floccular sludge were operated for aerobic granulation for 11 weeks. Changes in the phage, protozoa and bacterial communities were characterized throughout the granulation process. The filamentous phage, Inoviridae, increased in abundance at the initiation phase of granulation. However, the abundance shifted towards lytic phages during the maturation phase. In contrast, the abundance and diversity of protozoa decreased initially, possibly due to the reduction in settling time and subsequent washout. Upon the formation of granules, ciliated protozoa from the class Oligohymenophorea were the dominant group of protozoa based on metacommunity analysis. These protozoa had a strong, positive-correlation with the initial formation of compact aggregates prior to granule development. Furthermore, chemical inhibition of these ciliates in the floccular sludge delayed the initiation of granule formation. Analysis of the bacterial communities in the thiram treated sludge demonstrated that the recovery of 'Candidatus Accumulibacter' was positively correlated with the formation of compact aggregates and granules. CONCLUSION: Predation by bacteriophage and protozoa were positively correlated with the formation of aerobic granules. Increases in Inoviridae abundance suggested that filamentous phages may promote the structural formation of granules. Initiation of granules formation was delayed due to an absence of protozoa after chemical treatment. The presence of 'Candidatus Accumulibacter' was necessary for the formation of granules in the absence of protozoa.

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants.

BACKGROUND: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. RESULTS: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. CONCLUSIONS: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.

Sex, Scavengers, and Chaperones: Transcriptome Secrets of Divergent Symbiodinium Thermal Tolerances.

Corals rely on photosynthesis by their endosymbiotic dinoflagellates (Symbiodinium spp.) to form the basis of tropical coral reefs. High sea surface temperatures driven by climate change can trigger the loss of Symbiodinium from corals (coral bleaching), leading to declines in coral health. Different putative species (genetically distinct types) as well as conspecific populations of Symbiodinium can confer differing levels of thermal tolerance to their coral host, but the genes that govern dinoflagellate thermal tolerance are unknown. Here we show physiological and transcriptional responses to heat stress by a thermo-sensitive (physiologically susceptible at 32 °C) type C1 Symbiodinium population and a thermo-tolerant (physiologically healthy at 32 °C) type C1 Symbiodinium population. After nine days at 32 °C, neither population exhibited physiological stress, but both displayed up-regulation of meiosis genes by ≥ 4-fold and enrichment of meiosis functional gene groups, which promote adaptation. After 13 days at 32 °C, the thermo-sensitive population suffered a significant decrease in photosynthetic efficiency and increase in reactive oxygen species (ROS) leakage from its cells, whereas the thermo-tolerant population showed no signs of physiological stress. Correspondingly, only the thermo-tolerant population demonstrated up-regulation of a range of ROS scavenging and molecular chaperone genes by ≥ 4-fold and enrichment of ROS scavenging and protein-folding functional gene groups. The physiological and transcriptional responses of the Symbiodinium populations to heat stress directly correlate with the bleaching susceptibilities of corals that harbored these same Symbiodinium populations. Thus, our study provides novel, foundational insights into the molecular basis of dinoflagellate thermal tolerance and coral bleaching.

Characterization of the archaeal community fouling a membrane bioreactor.

Biofilm formation, one of the primary causes of biofouling, results in reduced membrane flux or increased transmembrane pressure and thus represents a major impediment to the wider implementation of membrane bioreactor (MBR) technologies for water purification. Most studies have focused on the role of bacteria in membrane fouling as they are the most dominant and best studied organisms present in the MBR. In contrast, there is limited information on the role of the archaeal community in biofilm formation in MBRs. This study investigated the composition of the archaeal community during the process of biofouling in an MBR. The archaeal community was observed to have lower richness and diversity in the biofilm than the sludge during the establishment of biofilms at low transmembrane pressure (TMP). Clustering of the communities based on the Bray-Curtis similarity matrix indicated that a subset of the sludge archaeal community formed the initial biofilms. The archaeal community in the biofilm was mainly composed of Thermoprotei, Thermoplasmata, Thermococci, Methanopyri, Methanomicrobia and Halobacteria. Among them, the Thermoprotei and Thermoplasmata were present at higher relative proportions in the biofilms than they were in the sludge. Additionally, the Thermoprotei, Thermoplasmata and Thermococci were the dominant organisms detected in the initial biofilms at low TMP, while as the TMP increased, the Methanopyri, Methanomicrobia, Aciduliprofundum and Halobacteria were present at higher abundances in the biofilms at high TMP.

The correlation between biofilm biopolymer composition and membrane fouling in submerged membrane bioreactors.

Biofouling, the combined effect of microorganism and biopolymer accumulation, significantly reduces the process efficiency of membrane bioreactors (MBRs). Here, four biofilm components, alpha-polysaccharides, beta-polysaccharides, proteins and microorganisms, were quantified in MBRs. The biomass of each component was positively correlated with the transmembrane pressure increase in MBRs. Proteins were the most abundant biopolymer in biofilms and showed the fastest rate of increase. The spatial distribution and co-localization analysis of the biofouling components indicated at least 60% of the extracellular polysaccharide (EPS) components were associated with the microbial cells when the transmembrane pressure (TMP) entered the jump phase, suggesting that the EPS components were either secreted by the biofilm cells or that the deposition of these components facilitated biofilm formation. It is suggested that biofilm formation and the accumulation of EPS are intrinsically coupled, resulting in biofouling and loss of system performance. Therefore, strategies that control biofilm formation on membranes may result in a significant improvement of MBR performance.

Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities.

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1-10 µm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28 days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4 µm PDMS surfaces. Also, field deployments over 28 days showed a significant reduction in biovolume on the 4 and 10 µm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10 µm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.

Predation response of Vibrio fischeri biofilms to bacterivorus protists.

Vibrio fischeri proliferates in a sessile, stable community known as a biofilm, which is one alternative survival strategy of its life cycle. Although this survival strategy provides adequate protection from abiotic factors, marine biofilms are still susceptible to grazing by bacteria-consuming protozoa. Subsequently, grazing pressure can be controlled by certain defense mechanisms that confer higher biofilm antipredator fitness. In the present work, we hypothesized that V. fischeri exhibits an antipredator fitness behavior while forming biofilms. Different predators representing commonly found species in aquatic populations were examined, including the flagellates Rhynchomonas nasuta and Neobodo designis (early biofilm feeders) and the ciliate Tetrahymena pyriformis (late biofilm grazer). V. fischeri biofilms included isolates from both seawater and squid hosts (Euprymna and Sepiola species). Our results demonstrate inhibition of predation by biofilms, specifically, isolates from seawater. Additionally, antiprotozoan behavior was observed to be higher in late biofilms, particularly toward the ciliate T. pyriformis; however, inhibitory effects were found to be widespread among all isolates tested. These results provide an alternative explanation for the adaptive advantage and persistence of V. fischeri biofilms and provide an important contribution to the understanding of defensive mechanisms that exist in the out-of-host environment.

The rise of pathogens: predation as a factor driving the evolution of human pathogens in the environment.

Bacteria in the environment must survive predation from bacteriophage, heterotrophic protists, and predatory bacteria. This selective pressure has resulted in the evolution of a variety of defense mechanisms, which can also function as virulence factors. Here we discuss the potential dual function of some of the mechanisms, which protect against heterotrophic protists, and how predation pressure leads to the evolution of pathogenicity. This is in accordance with the coincidental evolution hypothesis, which suggests that virulence factors arose as a response to other selective pressures, for example, predation rather than for virulence per se. In this review we discuss some of those environmental factors that may be associated with the rise of pathogens in the marine environment. In particular, we will discuss the role of heterotrophic protists in the evolution of virulence factors in marine bacteria. Finally, we will discuss the implications for expansion of current pathogens and emergence of new pathogens.

Optimal dosing regimen of nitric oxide donor compounds for the reduction of Pseudomonas aeruginosa biofilm and isolates from wastewater membranes.

Membrane fouling by bacterial biofilms remains a key challenge for membrane-based water purification systems. Here, the optimal biofilm dispersal potential of three nitric oxide (NO) donor compounds, viz. sodium nitroprusside, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (MAHMA NONOate) and 1-(hydroxy-NNO-azoxy)-L-proline, disodium salt, was investigated using Pseudomonas aeruginosa PAO1 as a model organism. Dispersal was quantitatively assessed by confocal microscopy [bacterial cells and the components of the extracellular polymeric substances (EPS) (polysaccharides and extracellular DNA)] and colony-forming unit counts. The three NO donor compounds had different optimal exposure times and concentrations, with MAHMA NONOate being the optimal NO donor compound. Biofilm dispersal correlated with a reduction in both bacterial cells and EPS. MAHMA NONOate also reduced single species biofilms formed by bacteria isolated from industrial membrane bioreactor and reverse osmosis membranes, as well as in isolates combined to generate mixed species biofilms. The data present strong evidence for the application of these NO donor compounds for prevention of biofouling in an industrial setting.

Dynamics of biofilm formation under different nutrient levels and the effect on biofouling of a reverse osmosis membrane system.

Pseudomonas aeruginosa PAO1 wild type and a mucoid derivative (FRD1) which over produces alginate were used to foul reverse osmosis (RO) membranes. When operated at a constant flux, biofilm formation on the RO membrane resulted in a slow rise in transmembrane pressure (TMP) of 22% for the initial four days of operation, followed by a sharp increase of 159% over the following two days. The initial slow increase in TMP was probably due to the formation of a biofilm on the membrane surface, which then accelerated the rate of biofouling through the effect of concentration polarization. At later stages of operation, most of the bacterial biomass consisted of dead cells. The amount of extracellular polymeric substances appeared to correlate positively with the number of dead cells. The results indicate that prolonging the initial stage of slow TMP increase and avoiding the latter stage of accelerated TMP increase would provide a sustainable operation of the RO system. These results suggest that nutrient limitation could reduce biofilm accumulation and delay the increase in TMP.

Protozoan predation as a driver of diversity and virulence in bacterial biofilms.

Protozoa are eukaryotic organisms that play a crucial role in nutrient cycling and maintaining balance in the food web. Predation, symbiosis and parasitism are three types of interactions between protozoa and bacteria. However, not all bacterial species are equally susceptible to protozoan predation as many are capable of defending against predation in numerous ways and may even establish either a symbiotic or parasitic life-style. Biofilm formation is one such mechanism by which bacteria can survive predation. Structural and chemical components of biofilms enhance resistance to predation compared to their planktonic counterparts. Predation on biofilms gives rise to phenotypic and genetic heterogeneity in prey that leads to trade-offs in virulence in other eukaryotes. Recent advances, using molecular and genomics techniques, allow us to generate new information about the interactions of protozoa and biofilms of prey bacteria. This review presents the current state of the field on impacts of protozoan predation on biofilms. We provide an overview of newly gathered insights into (i) molecular mechanisms of predation resistance in biofilms, (ii) phenotypic and genetic diversification of prey bacteria, and (iii) evolution of virulence as a consequence of protozoan predation on biofilms.

Chemically Mediated Interactions with Macroalgae Negatively Affect Coral Health but Induce Limited Changes in Coral Microbiomes.

Allelopathic chemicals facilitated by the direct contact of macroalgae with corals are potentially an important mechanism mediating coral-macroalgal interactions, but only a few studies have explored their impacts on coral health and microbiomes and the coral's ability to recover. We conducted a field experiment on an equatorial urbanized reef to assess the allelopathic effects of four macroalgal species (Bryopsis sp., Endosiphonia horrida, Hypnea pannosa and Lobophora challengeriae) on the health and microbiomes of three coral species (Merulina ampliata, Montipora stellata and Pocillopora acuta). Following 24 h of exposure, crude extracts of all four macroalgal species caused significant coral tissue bleaching and reduction in effective quantum yield. The corals were able to recover within 72 h of the removal of extracts, except those that were exposed to L. challengeriae. While some macroalgal extracts caused an increase in the alpha diversity of coral microbiomes, there were no significant differences in the composition and variability of coral microbiomes between controls and macroalgal extracts at each sampling time point. Nevertheless, DESeq2 differential abundance analyses showed species-specific responses of coral microbiomes. Overall, our findings provide insights on the limited effect of chemically mediated interactions with macroalgae on coral microbiomes and the capacity of corals to recover quickly from the macroalgal chemicals.