Vibrio cholerae is a ubiquitous marine bacterium that solubilizes and consumes chitin in the marine water column. In both the marine environment and the intestinal tract, V. cholerae forms biofilms: how do the diverse surfaces that V. cholerae encounters influence its biofilm formation and, in turn, shape its ecological interactions with other microbes? Here, we use the interaction between the predator Bdellovibrio bacteriovorus and V. cholerae as a model to explore how the environmental chitin substrate alters V. cholerae biofilm formation and predator-prey dynamics. We find that glass-bound biofilm growth provides strong protection for V. cholerae against predation while also allowing a population of predatory B. bacteriovorus to remain in place among prey cells. In contrast, chitin-bound biofilm structure offers less protection against B. bacteriovorus predation and does not maintain as stable a population of B. bacteriovorus. Using percolation and population dynamics models, we predict that these changes in predator-prey dynamics can be explained largely by alterations in V. cholerae biofilm architecture between the two conditions, which changes the fraction of prey available to B. bacteriovorus. Using targeted biofilm matrix gene deletions, we confirm this prediction by recapitulating key features of the chitin predator-prey interactions on glass surfaces. Following on this observation, we show that V. cholerae biofilms grown on chitin produce much less of the canonical biofilm matrix components and instead rely on other extracellular structures. Overall, our experiments detail how growth substrate can alter biofilm matrix composition and how these changes in biofilm architecture impact higher-order ecological interactions.

Purpose of review The growing threat of antibiotic-resistant pathogens, particularly in ocular infections like bacterial keratitis, necessitates alternative therapeutic strategies. This review evaluates the potential therapeutic role of predatory bacteria as novel live antimicrobials, offering a timely exploration of their potential in overcoming resistance mechanisms such as biofilm formation and persister cell development. Recent findings Predatory bacteria, including Bdellovibrio bacterovorus and Micavibrio aerguinosavorus selectively target Gram-negative bacteria, including Pseudomonas aeruginosa , while sparing Gram-positive ocular surface. They exhibit rapid bactericidal activity and efficacy against biofilms, persister cells, and antibiotic-resistant pathogens, but induce little inflammation. Advances in storage and delivery methods, such as lyophilization, cryomicroneedles, and thermoresponsive hydrogels, have potential to increase their therapeutic feasibility. However, in-vivo efficacy remains variable and their narrow spectrum limits effectiveness against Gram-positive pathogens. Summary Predatory bacteria present a promising alternative to traditional antibiotics in ocular therapeutics, particularly for drug-resistant infections. Integration of predatory bacteria with bacteriophages or conventional antibiotics may further optimize their potential. Continued translational research is essential to address current limitation and to validate their safety and efficacy for human or veterinary applications.

Vibrio cholerae is a ubiquitous marine microbe that solubilizes and consumes chitin in the marine water column. In both the marine environment and the intestinal track, V. cholerae forms biofilms; a key question regarding the lifestyle of V. cholerae is how do the diverse substrates that it encounters influence its biofilm formation and, in turn, shape its ecological interactions. Here, we use the predator-prey interaction between Bdellovibrio bacteriovorus and V. cholerae as a model to explore how the environmental chitin substrate alters V. cholerae biofilm formation and predator-prey interactions. We find that glass-bound biofilms provide strong protection for V. cholerae against predation while also allowing a population of predatory B. bacteriovorus to remain in place. In contrast, chitin-bound biofilms offer less protection against B. bacteriovorus predation and do not maintain a stable population of B. bacteriovorus. Using percolation and population dynamics models, we predict that these changes in predator-prey dynamics can be mostly explained by alterations in biofilm architecture between the two conditions, which changes the fraction of prey available to B. bacteriovorus. Performing targeted biofilm matrix deletions, we confirm this prediction by recapitulating key features of the chitin predator-prey interactions on glass surfaces. Following on this observation, we show that V. cholerae biofilms grown on chitin produce much less of the canonical biofilm matrix components and instead rely on other extracellular structures. Overall, our experiments detail how growth substrate can alter biofilm matrix composition and how these changes in biofilm architecture and cellular arrangement can impact higher-order ecological interactions.

Bdellovibrio bacteriovorus is the most studied member of a group of small motile Gram-negative bacteria called Bdellovibrio and Like Organisms (BALOs). B. bacteriovorus can prey on Gram-negative bacteria, including multi-drug resistant pathogens, and has been proposed as an alternative to antibiotics. Although the life cycle of B. bacteriovorus is well characterized, some molecular aspects of B. bacteriovorus-prey interaction are poorly understood. Hypothetical proteins with unestablished functions have been implicated in B. bacteriovorus predation by many studies. Our approach to characterize these proteins employing Alphafold has revealed novel interactions among attack phase-hypothetical proteins, which may be involved in less understood mechanisms of the Bdellovibrio attack phase. Here, we overlapped attack phase genes from B. bacteriovorus transcriptomic data sets and from transposon sequencing data sets to generate a set of proteins that are both expressed at the attack phase and are necessary for predation, which we termed Attack Phase Predation-Essential Proteins (AP-PEP). By applying Markov Cluster Algorithm and AlphaFold-Multimer to analyze the protein network and interaction partners of the AP-PEPs, we predicted high-confidence protein-protein interactions and two structurally similar but unique novel protein complexes formed among proteins of the Bd2209-Bd2212 and Bd2723-Bd2726 operons. Furthermore, we confirmed the interaction between hypothetical proteins Bd0075 and Bd0474 using the Bacteria Adenylate Cyclase Two-Hybrid system. In addition, we confirmed that the C-terminal domain of Bd0075, which contains Tetratricopeptide repeat motifs, participates principally in its interaction with Bd0474. This study revealed previously unknown cooperation among predation essential hypothetical proteins in the attack phase B. bacteriovorus and has paved the way for further work to understand molecular mechanisms of BALO predation processes.

The increase in multi-drug resistant (MDR) pathogens and the decline in the number of new antibiotics in the production pipeline pose a serious threat to our ability to treat infectious diseases. In this new landscape, once treatable diseases are now potentially life-threatening. This impending danger requires that urgent attention should be given to developing alternative strategies for combating MDR bacteria. A novel alternative is the use of predatory bacteria, B. bacteriovorus spp, that naturally prey on Gram-negative bacteria, including MDR Enterobacteriaceae. B. bacteriovorus has been shown to be non-pathogenic in animal models and on human cell lines, supporting its feasibility to be used to treat infections in animals and possibly humans. This document reviews various aspects of B. bacteriovorus biology, including its unique life cycle, "predatory toolbox", prey range, and recent research advances exploring B. bacteriovorus as an antimicrobial agent, stepping towards its use in human therapy. We also discuss the advantages and limitations of using B. bacteriovorus therapy and the strategies to overcome these limitations.

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand white rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and to a lesser extent S. marcescens. However, it was not able to significantly reduce the number of intraocular S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa and S. marcescens requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data indicate in vivo inhibition of Gram-negative bacteria proliferation within the intra-ocular environment by predatory bacteria.

AbstractEndogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth ofPseudomonas aeruginosa, Serratia marcescens, andStaphylococcus aureuswas evaluated in a New Zealand White rabbit endophthalmitis prevention model. Predatory bacteriaBdellovibrio bacteriovorusandMicavibrio aeruginosavoruswere able to reduce proliferation of keratitis isolates ofP. aeruginosaandS. marcescens. However, it was not able to significantly reduceS. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect onP. aeruginosarequires active predation rather than an antimicrobial immune response. Similarly, UV-inactivatedB. bacteriovoruswere unable to prevent proliferation ofP. aeruginosa. Together, these data suggestin vivopredation of Gram-negative bacteria within the intra-ocular environment.

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand White rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and S. marcescens. However, it was not able to significantly reduce S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data suggest in vivo predation of Gram-negative bacteria within the intra-ocular environment.

Purpose Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. Methods Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic Δ lasR mutant and co-injected with PBS or B. bacteriovorus . After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. Results We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The Δ lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus . Conclusion These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

Adherence to surfaces and secretion of extracellular matrix, or biofilm formation, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacterium Bdellovibrio bacteriovorus . This predator can kill and consume many different Gram-negative bacteria, including Vibrio cholerae and Escherichia coli . V. cholerae can protect itself from predation within highly packed biofilm structures that it creates, whereas E. coli biofilms are highly susceptible to B. bacteriovorus . Here we explore how predator-prey dynamics change when V. cholerae and E. coli are growing in biofilms together. We find that in dual species prey biofilms, E. coli survival under B. bacteriovorus predation increases, whereas V. cholerae survival decreases. E. coli benefits from predator protection when it becomes embedded within expanding groups of highly packed V. cholerae . But we also find that the ordered, highly packed, and clonal biofilm structure of V. cholerae can be disrupted if V. cholerae cells are directly adjacent to E. coli cells at the start of biofilm growth. When this occurs, the two species become entangled, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure to B. bacteriovorus . Significance Statement Bacteria live in multispecies, spatially structured communities ubiquitously in the natural world. These communities, or biofilms, have a strong impact on microbial ecology, but we often do not know how cellular scale interactions determine overall biofilm structure and community dynamics. Here we explore this problem in the context of predator-prey interaction, with two prey species – Vibrio cholerae and Escherichia coli – being attacked by the bacterial predator Bdellovibrio bacteriovorus . We find that when V. cholerae and E. coli grow together in biofilms, the architectures that they both produce change in ways that cannot be predicted from looking at each prey species alone, and that these changes in cell group structure impact the community dynamics of predator-prey interaction in biofilms.

The predatory bacterium Bdellovibrio bacteriovorus follows a life cycle in which it attaches to the exterior of a Gram-negative prey cell, enters the periplasm, and harvests resources to replicate before lysing the host to find new prey. Predatory bacteria such as this are common in many natural environments, as are groups of matrix-bound clusters of prey cells, termed biofilms. Despite the ubiquity of both predatory bacteria and biofilm-dwelling prey, the interaction between B. bacteriovorus and prey cells inside biofilms has received little attention and has not yet been studied at the micrometer scale. Filling this knowledge is critical to understanding the nature of predator-prey interaction in nature. Here we show that B. bacteriovorus is able to prey upon biofilms of the pathogen Vibrio cholerae , but only up until a critical maturation threshold past which the prey biofilms are protected from their predators. We determine the contribution of matrix secretion and cell-cell packing of the prey biofilm toward this protection mechanism. Our results demonstrate that B. bacteriovorus predation in the context of this protection threshold fundamentally transforms the sub-millimeter scale landscape of biofilm growth, as well as the process of community assembly as new potential biofilm residents enter the system.

It was previously demonstrated that predatory bacteria are able to efficiently eliminate Gram-negative pathogens including antibiotic-resistant and biofilm-associated bacteria. In this proof-of-concept study we evaluated whether two species of predatory bacteria, Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus, were able to alter the survival of Gram-negative pathogens on the ocular surface. Clinical keratitis isolates of Pseudomonas aeruginosa (strain PAC) and Serratia marcescens (strain K904) were applied to the ocular surface of NZW rabbits followed by application of predatory bacteria. At time intervals, surviving pathogenic bacteria were enumerated. In addition, B. bacteriovorus and S. marcescens were applied to porcine organ culture corneas under contact lenses, and the ocular surface was examined by scanning electron microscopy. The ocular surface epithelial layer of porcine corneas exposed to S. marcescens, but not B. bacteriovorus was damaged. Using this model, neither pathogen could survive on the rabbit ocular surface for longer than 24 h. M. aeruginosavorus correlated with a more rapid clearance of P. aeruginosa but not S. marcescens from rabbit eyes. This study supports previous evidence that predatory bacteria are well tolerated by the cornea, but suggest that predatory bacteria do not considerably change the ability of the ocular surface to clear the tested Gram-negative bacterial pathogens from the ocular surface.

Bdellovibrio and like organisms (BALOs) are obligate predatory bacteria commonly encountered in the environment. In dual predator-prey cultures, prey accessibility ensures optimal feeding and replication and rapid BALO population growth. However, the environmental prey landscape is complex, as it also incorporates non-prey cells and other particles. These may act as decoys, generating unproductive encounters which in turn may affect both predator and prey population dynamics. In this study, we hypothesized that increasing decoy:prey ratios would bring about increasing costs on the predator's reproductive fitness. We also tested the hypothesis that different BALOs and decoys would have different effects. To this end, we constructed prey landscapes including periplasmic or epibiotic predators including two types of decoy under a large range of initial decoy:prey ratio, and mixed cultures containing multiple predators and prey. We show that as decoy:prey ratios increase, the maximal predator population sizes is reduced and the time to reach it significantly increases. We found that BALOs spent less time handling non-prey (including superinfection-immune invaded prey) than prey cells, and did not differentiate between efficient and less efficient prey. This may explain why in multiple predator and prey cultures, less preferred prey appear to act as decoy.

The small size of bacterial cells necessitates rapid adaption to sudden environmental changes. In Bdellovibrio bacteriovorus, an obligate predator of bacteria common in oligotrophic environments, the non-replicative, highly motile attack phase (AP) cell must invade a prey to ensure replication. AP cells swim fast and respire at high rates, rapidly consuming their own contents. How the predator survives in the absence of prey is unknown. We show that starvation for prey significantly alters swimming patterns and causes exponential decay in prey-searching cells over hours, until population-wide swim-arrest. Swim-arrest is accompanied by changes in energy metabolism, enabling rapid swim-reactivation upon introduction of prey or nutrients, and a sweeping change in gene expression and gene regulation that largely differs from those of the paradigmatic stationary phase. Swim-arrest is costly as it imposes a fitness penalty in the form of delayed growth. We track the control of the swim arrest-reactivation process to cyclic-di-GMP (CdG) effectors, including two motility brakes. CRISPRi transcriptional inactivation, and in situ localization of the brakes to the cell pole, demonstrated their essential role for effective survival under prey-induced starvation. Thus, obligate predators evolved a unique CdG-controlled survival strategy, enabling them to sustain their uncommon lifestyle under fluctuating prey supply.

Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting.IMPORTANCEBdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.

Bdellovibrio and like organisms (BALOs) are largely distributed in soils and in water bodies obligate predators of gram-negative bacteria that can affect bacterial communities. Potential applications of BALOs include biomass reduction, their use against pathogenic bacteria in agriculture, and in medicine as an alternative against antibiotic-resistant pathogens. Such different environments and uses mean that BALOs should be active under a range of viscosities. In this study, the predatory behaviour of two strains of the periplasmic predator B. bacteriovorus and of the epibiotic predator Micavibrio aeruginosavorus was examined in viscous polyvinylpyrrolidone (PVP) solutions at 28 and at 37°C, using fluorescent markers and plate counts to track predator growth and prey decay. We found that at high viscosities, although swimming speed was largely decreased, the three predators reduced prey to levels similar to those of non-viscous suspensions, albeit with short delays. Prey motility and clumping did not affect the outcome. Strikingly, under low initial predator concentrations, predation dynamics were faster with increasing viscosity, an effect that dissipated with increasing predator concentrations. Changes in swimming patterns and in futile predator-predator encounters with viscosity, as revealed by path analysis under changing viscosities, along with possible PVP-mediated crowding effects, may explain the observed phenomena.

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans, Chryseobacterium gleum, Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis, but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum/S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans. These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

Multi-drug resistant bacterial infections are a serious threat to global public health. Changes in treatment modalities and prudent use of antibiotics can assist in reducing the threat, but new approaches are also required for untreatable cases. The use of predatory bacteria, such as Bdellovibrio bacteriovorus, is among the novel approaches being considered as possible therapeutics for antibiotic resistant and/or unidentified bacterial infections. Previous studies have examined the feasibility of using predatory bacteria to reduce colony-forming units (CFUs) in the lungs of rats exposed to lethal doses of Klebsiella pneumoniae; here we apply the approach to the Tier 1 select agent Yersinia pestis, and show that three doses of B. bacteriovorus introduced every six hours reduces the number of CFUs of Y. pestis in the lungs of inoculated mice by 86% after 24 h of infection. These experiments further demonstrate that predatory bacteria may serve to combat Gram negative bacterial infections, including those considered potential bioweapon agents, in the future.

The increase in multidrug-resistant Gram-negative bacterial infections has forced the reintroduction of antibiotics such as colistin. However, the spread of the plasmid-borne mcr-1 colistin resistance gene have moved us closer to an era of untreatable Gram-negative infections. To evaluate whether predatory bacteria could be used as a potential therapeutic to treat this upcoming threat, the ability of Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus to prey on several clinically relevant mcr-1-positive, colistin-resistant isolates was evaluated. No change in the ability of the predators to prey on free swimming and biofilms of prey cells harboring mcr-1 was measured, as compared to their mcr-1 negative strain.