Bdellovibrio's prey-independent lifestyle is fueled by amino acids as a carbon source.

Identifying the nutritional requirements and growth conditions of microorganisms is crucial for determining their applicability in industry and understanding their role in clinical ecology. Predatory bacteria such as Bdellovibrio bacteriovorus have emerged as promising tools for combating infections by human bacterial pathogens due to their natural killing features. Bdellovibrio's lifecycle occurs inside prey cells, using the cytoplasm as a source of nutrients and energy. However, this lifecycle supposes a challenge when determining the specific uptake of metabolites from the prey to complete the growth inside cells, a process that has not been completely elucidated. Here, following a model-based approach, we illuminate the ability of B. bacteriovorus to replicate DNA, increase biomass, and generate adenosine triphosphate (ATP) in an amino acid-based rich media in the absence of prey, keeping intact its predatory capacity. In this culture, we determined the main carbon sources used and their preference, being glutamate, serine, aspartate, isoleucine, and threonine. This study offers new insights into the role of predatory bacteria in natural environments and establishes the basis for developing new Bdellovibrio applications using appropriate metabolic and physiological methodologies. KEY POINTS: • Amino acids support axenic lifestyle of Bdellovibrio bacteriovorus. • B. bacteriovorus preserves its predatory ability when growing in the absence of prey.

Two-state swimming: Strategy and survival of a model bacterial predator in response to environmental cues.

Bdellovibrio bacteriovorus is a predatory bacterium preying upon Gram-negative bacteria. As such, B. bacteriovorus has the potential to control antibiotic-resistant pathogens and biofilm populations. To survive and reproduce, B. bacteriovorus must locate and infect a host cell. However, in the temporary absence of prey, it is largely unknown how B. bacteriovorus modulate their motility patterns in response to physical or chemical environmental cues to optimize their energy expenditure. To investigate B. bacteriovorus' predation strategy, we track and quantify their motion by measuring speed distributions as a function of starvation time. While an initial unimodal speed distribution relaxing to one for pure diffusion at long times may be expected, instead we observe a bimodal speed distribution with one mode centered around that expected from diffusion and the other centered at higher speeds. What is more, for an increasing amount of time over which B. bacteriovorus is starved, we observe a progressive reweighting from the active swimming state to an apparent diffusive state in the speed distribution. Distributions of trajectory-averaged speeds for B. bacteriovorus are largely unimodal, indicating switching between a faster swim speed and an apparent diffusive state within individual observed trajectories rather than there being distinct active swimming and apparent diffusive populations. We also find that B. bacteriovorus' apparent diffusive state is not merely caused by the diffusion of inviable bacteria as subsequent spiking experiments show that bacteria can be resuscitated and bimodality restored. Indeed, starved B. bacteriovorus may modulate the frequency and duration of active swimming as a means of balancing energy consumption and procurement. Our results thus point to a reweighting of the swimming frequency on a trajectory basis rather than a population level basis.

The effect of Bdellovibrio bacteriovorus containing dressing on superficial incisional surgical site infections experimentally induced by Klebsiella pneumoniae in mice.

Bdellovibrio bacteriovorus is a bacterial agent that stands out for its ability to act as a predator against gram-negative bacteria and has found application against antibiotic-resistant pathogens. The aim of this study is to determine the efficacy of Bdellovibrio bacteriovorus against antibiotic-resistant pathogens, particularly those causing infections in surgical incision sites. A total of 6 experimental groups were created in mice, and surgical area infections were initiated with Klebsiella pneumoniae in incision sites. The effects of antibiotics and Bdellovibrio bacteriovorus alone or in combination were compared to the control group. In the Bdellovibrio bacteriovorus treatment group, edema and redness were observed in all mice at 24th hours, in 20% of mice at 48th hours, and in none at the 72 nd h. A significant difference was observed in the Bdellovibrio bacteriovorus treatment groups in reducing Klebsiella pneumoniae burden in the incision area compared to antibiotics alone or Bdellovibrio bacteriovorus + antibiotics, (p < 0.001). Likewise, cytokine level determinations indicated that B. bacteriovorus applications generated a therapeutic response without inducing an inflammatory response.

Predator Versus Pathogen: How Does Predatory Bdellovibrio bacteriovorus Interface with the Challenges of Killing Gram-Negative Pathogens in a Host Setting?

Bdellovibrio bacteriovorus is a small deltaproteobacterial predator that has evolved to invade, reseal, kill, and digest other gram-negative bacteria in soils and water environments. It has a broad host range and kills many antibiotic-resistant, clinical pathogens in vitro, a potentially useful capability if it could be translated to a clinical setting. We review relevant mechanisms of B. bacteriovorus predation and the physiological properties that would influence its survival in a mammalian host. Bacterial pathogens increasingly display conventional antibiotic resistance by expressing and varying surface and soluble biomolecules. Predators coevolved alongside prey bacteria and so encode diverse predatory enzymes that are hard for pathogens to resist by simple mutation. Predators do not replicate outside pathogens and thus express few transport proteins and thus few surface epitopes for host immune recognition. We explain these features, relating them to the potential of predatory bacteria as cellular medicines.

Microbe Profile: Bdellovibrio bacteriovorus: a specialized bacterial predator of bacteria.

Bdellovibrio bacteriovorus is an environmentally-ubiquitous bacterium that uses unique adaptations to kill other bacteria. The best-characterized strain, HD100, has a multistage lifestyle, with both a free-living attack phase and an intraperiplasmic growth and division phase inside the prey cell. Advances in understanding the basic biology and regulation of predation processes are paving the way for future potential therapeutic and bioremediation applications of this unusual bacterium.

Predatory bacteria as living antibiotics - where are we now?

Antimicrobial resistance (AMR) is a global health and economic crisis. With too few antibiotics in development to meet current and anticipated needs, there is a critical need for new therapies to treat Gram-negative infections. One potential approach is the use of living predatory bacteria, such as Bdellovibrio bacteriovorus (small Gram-negative bacteria that naturally invade and kill Gram-negative pathogens of humans, animals and plants). Moving toward the use of Bdellovibrio as a 'living antibiotic' demands the investigation and characterization of these bacterial predators in biologically relevant systems. We review the fundamental science supporting the feasibility of predatory bacteria as alternatives to antibiotics.

Bdellovibrio bacteriovorus: a potential 'living antibiotic' to control bacterial pathogens.

Bdellovibrio bacteriovorus is a small Deltaproteobacterium which, since its discovery, has distinguished itself for the unique ability to prey on other Gram-negative bacteria. The studies on this particular "predatory bacterium", have gained momentum in response to the rising problem of antibiotic resistance, because it could be applied as a potential probiotic and antibiotic agent. Hereby, we present recent advances in the study of B. bacteriovorus, comprehending fundamental aspects of its biology, obligatory intracellular life cycle, predation resistance, and potential applications. Furthermore, we discuss studies that pave the road towards the use of B. bacteriovorus as a "living antibiotic" in human therapy, focussing on its interaction with biofilms, the host immune response, predation susceptibility and in vivo application models. The available data imply that it will be possible to upgrade this predator bacterium from a predominantly academic interest to an instrument that could confront antibiotic resistant infections.

Predatory and biocontrol potency of Bdellovibrio bacteriovorus toward phytopathogenic strains of Pantoea sp. and Xanthomonas campestris in the presence of exo-biopolymers: in vitro and in vivo assessments.

Bdellovibrios are predatory bacteria that invade other live Gram-negative bacterial cells for growth and reproduction. They have recently been considered as potential living antibiotics and biocontrol agents. In this study, the predatory activity and biocontrol potency of Bdellovibrio bacteriovorus strain SOIR-1 against Pantoea sp. strain BCCS and Xanthomonas campestris, two exo-biopolymer-producing phytopathogens, was evaluated. Plaque formation assays and lysis analysis in the broth co-cultures were used for the in vitro evaluation of bacteriolytic activity of strain SOIR-1. The in vivo biocontrol potential of strain SOIR-1 was evaluated by pathogenicity tests on the onion bulbs and potato tuber slices. The phytopathogens were also recovered from the infected plant tissues and confirmed using biochemical tests and PCR-based 16S rRNA gene sequence analysis. Typical bdellovibrios plaques were developed on the lawn cultures of Pantoea sp. BCCS and X. campestris. The killing rate of strain SOIR-1 toward Pantoea sp. BCCS and X. campestris was 84.3% and 76.3%, respectively. Exo-biopolymers attenuated the predation efficiency of strain SOIR-1 up to 10.2-18.2% (Pantoea sp. BCCS) and 12.2-17.3% (X. campestris). The strain SOIR-1 significantly reduced rotting symptoms in the onion bulbs caused by Pantoea sp. BCCS (69.0%) and potato tuber slices caused by X. campestris (73.1%). Although more field assessments are necessary, strain SOIR-1 has the preliminary potential as a biocontrol agent against phytopathogenic Pantoea sp. BCCS and X. campestris, especially in postharvest storage. Due to the particular physicochemical properties of evaluated exo-biopolymers, they can be used in the designing encapsulation systems for delivery of bdellovibrios.

Diffusible Signaling Factor, a Quorum-Sensing Molecule, Interferes with and Is Toxic Towards Bdellovibrio bacteriovorus 109J.

Bdellovibrio bacteriovorus 109J is a predatory bacterium which lives by predating on other Gram-negative bacteria to obtain the nutrients it needs for replication and survival. Here, we evaluated the effects two classes of bacterial signaling molecules (acyl homoserine lactones (AHLs) and diffusible signaling factor (DSF)) have on B. bacteriovorus 109J behavior and viability. While AHLs had a non-significant impact on predation rates, DSF considerably delayed predation and bdelloplast lysis. Subsequent experiments showed that 50 µM DSF also reduced the motility of attack-phase B. bacteriovorus 109J cells by 50% (38.2 ± 14.9 vs. 17 ± 8.9 µm/s). Transcriptomic analyses found that DSF caused genome-wide changes in B. bacteriovorus 109J gene expression patterns during both the attack and intraperiplasmic phases, including the significant downregulation of the flagellum assembly genes and numerous serine protease genes. While the former accounts for the reduced speeds observed, the latter was confirmed experimentally with 50 µM DSF completely blocking protease secretion from attack-phase cells. Additional experiments found that 30% of the total cellular ATP was released into the supernatant when B. bacteriovorus 109J was exposed to 200 µM DSF, implying that this QS molecule negatively impacts membrane integrity.

Spatial heterogeneity stabilizes predator-prey interactions at the microscale while patch connectivity controls their outcome.

Natural landscapes are both fragmented and heterogeneous, affecting the distribution of organisms, and their interactions. While predation in homogeneous environments increases the probability of population extinction, fragmentation/heterogeneity promotes coexistence and enhances community stability as shown by experimentation with animals and microorganisms, and supported by theory. Patch connectivity can modulate such effects but how microbial predatory interactions are affected by water-driven connectivity is unknown. In soil, patch habitability by microorganisms, and their connectivity depend upon the water saturation degree (SD). Here, using the obligate bacterial predator Bdellovibrio bacteriovorus, and a Burkholderia prey, we show that soil spatial heterogeneity profoundly affects predatory dynamics, enhancing long-term co-existence of predator and prey in a SD-threshold dependent-manner. However, as patches and connectors cannot be distinguished in these soil matrices, metapopulations cannot be invoked to explain the dynamics of increased persistence. Using a set of experiments combined with statistical and physical models we demonstrate and quantify how under full connectivity, predation is independent of water content but depends on soil microstructure characteristics. In contrast, the SD below which predation is largely impaired corresponds to a threshold below which the water network collapses and water connectivity breaks down, preventing the bacteria to move within the soil matrix.

Viscosity has dichotomous effects on Bdellovibrio bacteriovorus HD100 predation.

Bdellovibrio bacteriovorus HD100 is a highly motile predatory bacterium that consumes other Gram-negative bacteria for its sustenance. Here, we describe the impacts the media viscosity has both on the motility of predator and its attack rates. Experiments performed in polyethylene glycol (PEG) solutions, a linear polymer, found a viscosity of 10 mPa s (5% PEG) negatively impacted predation over a 24-h period. When the viscosity was increased to 27 mPa s (10% PEG), predation was nearly abolished. Tests with three other B. bacteriovorus strains, i.e., 109J and two natural isolates, found identical results. Short-term (2-h) experiments, however, found attack rates were improved in 1% PEG, which had a viscosity of 5.4 mPa s, using bioluminescent prey and their viabilities. In contrast, when experiments were performed in dextran, a branched polymer, no increase in predation was seen even though the viscosity was a comparable 5.1 mPa s. The enhanced attack rates in this solution coincided with a 31% increase in B. bacteriovorus HD100 swimming speeds (62 µm s-1 in 1% PEG vs. 47.5 µm s-1 in HEPES-salt).

Dynamics of Chromosome Replication and Its Relationship to Predatory Attack Lifestyles in Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus degrades a host's macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus Our results show that in B. bacteriovorus, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if this B. bacteriovorus life cycle fails in some cells of Escherichia coli, they can instead use second prey cells to complete their life cycle.IMPORTANCE New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium Bdellovibrio bacteriovorus, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The B. bacteriovorus life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host's macromolecules and reuses them for its own growth. To understand the use of B. bacteriovorus as a "living antibiotic," it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell of B. bacteriovorus This process initiates at the invasion pole of B. bacteriovorus and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells sequentially to complete their life cycle.

Behaviour of Bdellovibrio bacteriovorus in the presence of Gram-positive Staphylococcus aureus.

The present study aimed to characterize the behavior of Bdellovibrio bacteriovorus in the presence of Staphylococcus aureus. B. bacteriovorus was co-cultured with S. aureus or Pseudomonas aeruginosa or Streptococcus mutans, in planktonic and sessile conditions. Co-cultures were studied by Field-Emission Scanning Electron Microscopy (FESEM), Scanning Transmission Electron Microscopy (STEM), turbidimetry, quantitative PCR (qPCR), and sequencing of gene Bd0108 of B. bacteriovorus. Results indicated that B. bacteriovorus comparably inhibited planktonic growth of P. aeruginosa and S. aureus, but not of S. mutans. FESEM and STEM showed that B. bacteriovorus interacts with S. aureus affecting its cell wall and membrane. Sequencing of gene Bd0108 did not reveal any of the mutations that can arise from the host-interaction (hit) locus. Although some Gram-negative species are reported to be B. bacteriovorus prey, it seems that in case of nutrient deficiency this predatory bacterium can also take advantage of some Gram-positive species. B. bacteriovorus behaviour in the presence of S. aureus is relevant for its possible therapeutic use in several pathologies, like cystic fibrosis in which S. aureus and P. aeruginosa frequently coexist as infectious agents.

Hydrodynamic Hunters.

The Gram-negative Bdellovibrio bacteriovorus (BV) is a model bacterial predator that hunts other bacteria and may serve as a living antibiotic. Despite over 50 years since its discovery, it is suggested that BV probably collides into its prey at random. It remains unclear to what degree, if any, BV uses chemical cues to target its prey. The targeted search problem by the predator for its prey in three dimensions is a difficult problem: it requires the predator to sensitively detect prey and forecast its mobile prey's future position on the basis of previously detected signal. Here instead we find that rather than chemically detecting prey, hydrodynamics forces BV into regions high in prey density, thereby improving its odds of a chance collision with prey and ultimately reducing BV's search space for prey. We do so by showing that BV's dynamics are strongly influenced by self-generated hydrodynamic flow fields forcing BV onto surfaces and, for large enough defects on surfaces, forcing BV in orbital motion around these defects. Key experimental controls and calculations recapitulate the hydrodynamic origin of these behaviors. While BV's prey (Escherichia coli) are too small to trap BV in hydrodynamic orbit, the prey are also susceptible to their own hydrodynamic fields, substantially confining them to surfaces and defects where mobile predator and prey density is now dramatically enhanced. Colocalization, driven by hydrodynamics, ultimately reduces BV's search space for prey from three to two dimensions (on surfaces) even down to a single dimension (around defects). We conclude that BV's search for individual prey remains random, as suggested in the literature, but confined, however-by generic hydrodynamic forces-to reduced dimensionality.

Biocontrol of Burkholderia cepacia complex bacteria and bacterial phytopathogens by Bdellovibrio bacteriovorus.

Bdellovibrio and like organisms are predatory bacteria that have the unusual property of using the cytoplasmic constituents of other Gram-negative bacteria as nutrients. These predators may thus provide an alternative approach to the biocontrol of human and plant pathogens. Predators were isolated on Burkholderia cenocepacia K56-2 and J2315 as prey cells, in enrichment cultures with soil and sewage. Three isolates (DM7C, DM8A, and DM11A) were identified as Bdellovibrio bacteriovorus on the basis of morphology, a periplasmic life cycle, and 16S rRNA gene sequencing. The prey range of these isolates was tested on Burkholderia cepacia complex bacteria and several phytopathogenic bacteria of agricultural importance. Of 31 strains of the Burkholderia cepacia complex tested, only 4 were resistant to predation by strain DM7C. A subset of 9 of the prey tested were also susceptible to strains DM8A and DM11A. Of 12 phytopathogens tested, 4 were resistant to strains DM7C and DM8A, and only 2 were resistant to strain DM11A. Thus, Bdellovibrio bacteriovorus strains retrieved from environmental samples on 2 Burkholderia cenocepacia isolates from cystic fibrosis patients did not distinguish in their prey range between other isolates of that pathogen or phytopathogens. Such strains hold promise as potential wide-spectrum biocontrol agents.

Isolation of Bdellovibrio bacteriovorus from a tropical wastewater treatment plant and predation of mixed species biofilms assembled by the native community members.

It is reported here that a predatory bacterium belonging to the Genus Bdellovibrio, was isolated from activated sludge at the Ulu Pandan Water Reclamation Plant, Singapore. 16S rDNA gene sequencing analysis revealed that this isolate was 99% identical to 'Bdellovibrio bacteriovorus strain Tiberius' and hence is designated as 'Bdellovibrio bacteriovorus UP'. Using a novel approach based on fluorescence in situ hybridization (FISH), a prey cell density-dependent growth pattern of B. bacteriovorus UP was established. B. bacteriovorus UP preyed upon a broad range of bacterial species (60 species) isolated from the activated sludge. Except for Ochrobactrum anthropi, all Gram-negative species were sensitive to predation by B. bacteriovorus UP irrespective of the mode of growth (planktonic or biofilm). Similarly, the predation-sensitive species were not protected by the predation-resistant species, O. anthropi, as determined in multiple dual-species planktonic and biofilm consortia. Given the broad prey spectrum, B. bacteriovorus UP may impact functional community members, which are largely members of the Proteobacteria. Thus, these results provide an important insight to the role of predatory bacteria in shaping of community structure and function in both natural and engineered ecosystems.

Flagellar stator genes control a trophic shift from obligate to facultative predation and biofilm formation in a bacterial predator.

The bacterial predator Bdellovibrio bacteriovorus is considered to be obligatorily prey (host)-dependent (H-D), and thus unable to form biofilms. However, spontaneous host-independent (H-I) variants grow axenically and can form robust biofilms. A screen of 350 H-I mutants revealed that single mutations in stator genes fliL or motA were sufficient to generate flagellar motility-defective H-I strains able to adhere to surfaces but unable to develop biofilms. The variants showed large transcriptional shifts in genes related to flagella, prey-invasion, and cyclic-di-GMP (CdG), as well as large changes in CdG cellular concentration relative to the H-D parent. The introduction of the parental fliL allele resulted in a full reversion to the H-D phenotype, but we propose that specific interactions between stator proteins prevented functional complementation by fliL paralogs. In contrast, specific mutations in a pilus-associated protein (Bd0108) mutant background were necessary for biofilm formation, including secretion of extracellular DNA (eDNA), proteins, and polysaccharides matrix components. Remarkably, fliL disruption strongly reduced biofilm development. All H-I variants grew similarly without prey, showed a strain-specific reduction in predatory ability in prey suspensions, but maintained similar high efficiency in prey biofilms. Population-wide allele sequencing suggested additional routes to host independence. Thus, stator and invasion pole-dependent signaling control the H-D and the H-I biofilm-forming phenotypes, with single mutations overriding prey requirements, and enabling shifts from obligate to facultative predation, with potential consequences on community dynamics. Our findings on the facility and variety of changes leading to facultative predation also challenge the concept of Bdellovibrio and like organisms being obligate predators. IMPORTANCE: The ability of bacteria to form biofilms is a central research theme in biology, medicine, and the environment. We show that cultures of the obligate (host-dependent) "solitary" predatory bacterium Bdellovibrio bacteriovorus, which cannot replicate without prey, can use various genetic routes to spontaneously yield host-independent (H-I) variants that grow axenically (as a single species, in the absence of prey) and exhibit various surface attachment phenotypes, including biofilm formation. These routes include single mutations in flagellar stator genes that affect biofilm formation, provoke motor instability and large motility defects, and disrupt cyclic-di-GMP intracellular signaling. H-I strains also exhibit reduced predatory efficiency in suspension but high efficiency in prey biofilms. These changes override the requirements for prey, enabling a shift from obligate to facultative predation, with potential consequences on community dynamics.

Biocontrol treatment: Application of Bdellovibrio bacteriovorus HD100 against burn wound infection caused by Pseudomonas aeroginosa in mice.

Owing to the high level of resistance to various antibiotics in bacteria causing burn wound infections, the alternative therapeutics is highly demanded. Bdellovibrio and like organisms (BALOs) seem to be a superb choice. In the present study, Bdellovibrio bacteriovorus HD100 was selected for treating burn wound infection caused by Pseudomonas aeruginosa strain PAO1 in a mouse model. In this experiment, two treatments, meropenem as antibiotic and B. bacteriovorus, were employed. Histopathology indicated an accelerated healing rate in both treatments in comparison with the control. Moreover, quantitative reverse transcription PCR (qRT-PCR) was applied to investigate the expression of tnf-α (tumor necrosis factor alpha), pdgf (platelet-derived growth factor), tgf-ß1 (transforming growth factor beta1), ifn-γ (interferon gamma), vegf (vascular endothelial group factor), and col1 (collagen type 1). The results demonstrated that treating burn wound areas with Bdellovibrio not only decrease the inflammatory phase period, but also may improve the characteristics of proliferative phases of wound healing. In addition, a significant difference was explored between the two treatment groups in the regulation of all genes, except for pdgf revealed a significant up regulation in both treatment groups. The results disclose that Bdellovibrio attenuates P. aeruginosa in burn wounds infections and improves the wound healing process.

An optimized workflow to measure bacterial predation in microplates.

The predatory bacterium Bdellovibrio bacteriovorus invades and proliferates inside other bacteria by non-binary division. Here we describe a fluorescence-based technique for the immediate evaluation of predator density independently of plaque formation, an optimized setup to monitor predation in microplates, and the CuRveR package to quantify both prey killing and predator proliferation dynamics. This protocol allows to assess the impact of mutations or chemicals on predation. CuRveR also constitutes a user-friendly tool to analyze growth or decay data unrelated to predation. For complete details on the use and execution of this profile, please refer to Kaljevic et al., 2021.

Validating Flow Cytometry as a Method for Quantifying Bdellovibrio Predatory Bacteria and Its Prey for Microbial Ecology.

Bdellovibrio bacteriovorus is a predatory, Gram-negative bacteria that feeds on many pathogenic bacteria and has been investigated as a possible solution for mitigating biofilms in different fields. The application depends on more fundamental ecological studies into the dynamics between Bdellovibrio and their prey. To do so requires an accurate, reliable, and, preferably rapid, way of enumerating the cells. Flow cytometry (FCM) is potentially a rapid, accurate, and inexpensive tool for this, but it has yet to be validated in the enumeration of Bdellovibrio. In this study, we developed a protocol to measure the number of Bdellovibrio in samples of various densities using FCM and compared the results with those of other methods: optical density (OD), PFU assay (PFU), and quantitative PCR (qPCR). We observed a strong correlation between values obtained using FCM and PFU (ρ = 0.923) and FCM and qPCR (ρ = 0.987). Compared to optical density there was a much weaker correlation (ρ = 0.784), which was to be expected given the well-documented uncertainty in converting optical density (OD) to cell numbers. The FCM protocol was further validated by demonstrating its ability to distinguish and count mixed populations of Bdellovibrio and the prey Pseudomonas. Thus, the accuracy of FCM as well as its speed and reproducibility make it a suitable alternative for measuring Bdellovibrio cell numbers, especially where many samples are required to capture the dynamics of predator-prey interactions. IMPORTANCE The rise of antibiotic resistance and the unwanted growth of bacteria is a universally growing problem. Predatory bacteria can be used as a biological alternative to antibiotics because they grow by feeding on other bacteria. To apply this effectively requires further study and a deeper understanding of the forces that drive a prey population to elimination. Initially, such studies require more reliable methods to count these cells. Flow cytometry (FCM) is potentially a rapid, accurate, and inexpensive tool for this, but it has yet to be validated for predatory bacteria. This study develops a protocol to count the predatory bacteria Bdellovibrio bacteriovorus and its Pseudomonas prey using FCM and compare the results with those of other methods, demonstrating its ability for studies into B. bacteriovorus predation dynamics. This could lead to the use of B. bacteriovorus for killing bacterial biofilms in fields, such as drinking water and agriculture.