Ocular Delivery of Predatory Bacteria with Cryomicroneedles Against Eye Infection.

The development of potent antibiotic alternatives with rapid bactericidal properties is of great importance in addressing the current antibiotic crisis. One representative example is the topical delivery of predatory bacteria to treat ocular bacterial infections. However, there is a lack of suitable methods for the delivery of predatory bacteria into ocular tissue. This work introduces cryomicroneedles (cryoMN) for the ocular delivery of predatory Bdellovibrio bacteriovorus (B. bacteriovorus) bacteria. The cryoMN patches are prepared by freezing B. bacteriovorus containing a cryoprotectant medium in a microneedle template. The viability of B. bacteriovorus in cryoMNs remains above 80% as found in long-term storage studies, and they successfully impede the growth of gram-negative bacteria in vitro or in a rodent eye infection model. The infection is significantly relieved by nearly six times through 2.5 days of treatment without substantial effects on the cornea thickness and morphology. This approach represents the safe and efficient delivery of new class of antimicrobial armamentarium to otherwise impermeable ocular surface and opens up new avenues for the treatment of ocular surface disorders.

Secretion systems play a critical role in resistance to predation by Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, a Gram-negative predatory bacterium belonging to the Bdellovibrio and like organisms (BALOs), predates on Gram-negative bacteria. BALO strains differ in prey range but so far, the genetic basis of resistance against BALO predation is hardly understood. We developed a loss-of-function approach to screen for sensitive mutants in a library of strain M6, a predation-resistant strain of the plant pathogen Acidovorax citrulli. The screen is based on tracking the growth of a B. bacteriovorus strain expressing the fluorescent reporter Tdtomato in mutant pools to reveal predation-sensitive variants. Two independent loci were identified in mutant strains exhibiting significant levels of susceptibility to the predator. Genes in the two loci were analysed using both protein sequence homology and protein structure modeling. Both were secretion-related proteins and thus associated to the bacterial cell wall. Successful complementation of gspK, a gene encoding for a minor pseudopilin protein confirmed the involvement of the type II secretion system in A. citrulli M6 resistance. This proof of concept study shows that our approach can identify key elements of the BALO-prey interaction, and it validates the hypothesis that mutational changes in a single gene can drastically impact prey resistance to BALO predation.

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.

Studies on Bd0934 and Bd3507, Two Secreted Nucleases from Bdellovibrio bacteriovorus, Reveal Sequential Release of Nucleases during the Predatory Cycle.

Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades and kills a broad range of Gram-negative prey cells, including human pathogens. Its potential therapeutic application has been the subject of increased research interest in recent years. However, an improved understanding of the fundamental molecular aspects of the predatory life cycle is crucial for developing this bacterium as a "living antibiotic." During intracellular growth, B. bacteriovorus secretes an arsenal of hydrolases, which digest the content of the host cell to provide growth nutrients for the predator, e.g., prey DNA is completely degraded by the nucleases. Here, we have, on a genetic and molecular level, characterized two secreted DNases from B. bacteriovorus, Bd0934 and Bd3507, and determined the temporal expression profile of other putative secreted nucleases. We conclude that Bd0934 and Bd3507 are likely a part of the predatosome but are not essential for the predation, host-independent growth, prey biofilm degradation, and self-biofilm formation. The detailed temporal expression analysis of genes encoding secreted nucleases revealed that these enzymes are produced in a sequential orchestrated manner. This work contributes to our understanding of the sequential breakdown of the prey nucleic acid by the nucleases secreted during the predatory life cycle of B. bacteriovorusIMPORTANCE Antibiotic resistance is a major global concern with few available new means to combat it. From a therapeutic perspective, predatory bacteria constitute an interesting tool. They not only eliminate the pathogen but also reduce the overall pool of antibiotic resistance genes through secretion of nucleases and complete degradation of exogenous DNA. Molecular knowledge of how these secreted DNases act will give us further insight into how antibiotic resistance, and the spread thereof, can be limited through the action of predatory bacteria.

A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance.

Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).

Expression of attack and growth phase genes of Bdellovibrio bacteriovorus in the presence of Gram-negative and Gram-positive prey.

The expression of attack phase (AP) and growth phase (GP) genes of Bdellovibrio bacteriovorus (B. bacteriovorus) was compared in the presence of Gram-negative [Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Enterococcus faecium (E. faecium)] prey, using relative quantitative polymerase chain reaction (relative qPCR) assays. The genes bd0108 (pili retraction/extrusion) and merRNA (massively expressed riboswitch RNA) were highly expressed in the AP cells [3.99- to 6.06-fold (E. coli), 3.91- to 7.05-fold (K. pneumoniae) and 2.91- to 7.30-fold (E. faecium)]. The fliC1 gene (flagella filament) was also expressed at a high level in the AP cells however, after 240 min of co-culture with E. faecium the expression of fliC1 remained low (at 0.759-fold), while in the presence of the Gram-negative prey fliC1 expression increased. Additionally, the GP genes bd0816 (peptidoglycan-modifying enzyme) and groES1 (chaperone protein) were not induced in the presence of E. faecium. However, they were expressed in the early GP and GP of B. bacteriovorus after exposure to the Gram-negative prey. It can thus be concluded that B. bacteriovorus senses the presence of potential prey when exposed to Gram-positive and Gram-negative bacteria, however the GP genes are not induced in co-culture with E. faecium. The results from this study thus indicate that B. bacteriovorus does not actively grow in the presence of E. faecium and the second predatory cue (induces active growth of B. bacteriovorus) is lacking when B. bacteriovorus is co-cultured with the Gram-positive prey.

High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus.

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.

Lyophilization of Bdellovibrio bacteriovorus 109J for Long-Term Storage.

Bdellovibrio bacteriovorus 109J is a Gram-negative predatory bacterium with obligate host dependency on other Gram-negative bacteria. This bacteriolytic predator collides with, enters, and establishes growth within the prey (host) periplasm, eventually lysing the prey cell wall to release fresh, motile B. bacteriovorus progeny. Laboratory maintenance of B. bacteriovorus has been previously described by other investigators. The protocols included in this unit deal with the technique required to lyophilize or freeze dry host-dependent B. bacteriovorus. This is an alternative means to frozen glycerol stocks for the long-term storage of B. bacteriovorus. It includes the cultivation process and methods to lyophilize B. bacteriovorus as well as recommended storage conditions. In addition, this unit provides insight on the formulation's shelf-life including the time to active culture after reviving lyophilized stocks of B. bacteriovorus following short-, medium-, and long-term storage. © 2017 by John Wiley & Sons, Inc.

Catch me if you can: dispersal and foraging of Bdellovibrio bacteriovorus 109J along mycelia.

To cope with heterogeneous environments and resource distributions, filamentous fungi have evolved a spatially extensive growth enabling their hyphae to penetrate air-water interfaces and pass through air-filled pores. Such mycelia are also known to act as dispersal networks for the mobilisation of bacteria ('fungal highways') and connection of microbial microhabitats. Hitherto, however, nothing is known about the effect of mycelia-based dispersal on interactions between bacterial predators and their prey and concomitant effects on biomass formation. We here hypothesise that mycelia enable the contact between predators and their prey and shape a prey's population. We investigated the impact of predation by Bdellovibrio bacteriovorus 109J on the growth of its potential prey Pseudomonas fluorescens LP6a in the presence of mycelia. Our data give evidence that hyphae increase the accessibility of the prey to B. bacteriovorus 109J and, hence, allow for efficient foraging and shaping of prey populations not seen in the absence of mycelia. To test our hypothesis tailored microbial landscapes were used for better reduction of emerging properties in complex systems. Our data suggest that mycelia have substantial influence on prey-predator relationship and hereby may promote the structure of prey and predator populations and, hence, may be a determinant for biomass formation in heterogeneous environments.

Predatory bacteria are nontoxic to the rabbit ocular surface.

Given the increasing emergence of antimicrobial resistant microbes and the near absent development of new antibiotic classes, innovative new therapeutic approaches to address this global problem are necessary. The use of predatory bacteria, bacteria that prey upon other bacteria, is gaining interest as an "out of the box" therapeutic treatment for multidrug resistant pathogenic bacterial infections. Before a new antimicrobial agent is used to treat infections, it must be tested for safety. The goal of this study was to test the tolerability of bacteria on the ocular surface using in vitro and in vivo models. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were found to be non-toxic to human corneal stromal keratocytes in vitro; however, they did induce production of the proinflammatory chemokine IL-8 but not IL-1ß. Predatory bacteria did not induce inflammation on the ocular surface of rabbit eyes, with and without corneal epithelial abrasions. Unlike a standard of care antibiotic vancomycin, predatory bacteria did not inhibit corneal epithelial wound healing or increase clinical inflammatory signs in vivo. Together these data support the safety of predatory bacteria on the ocular surface, but future studies are warranted regarding the use predatory bacteria in deeper tissues of the eye.

Identification and differential production of ubiquinone-8 in the bacterial predator Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus 109J, a predatory bacterium with potential as a bacterial control agent, can exist in several lifestyles that differ both in predatory capacity and color. We determined that levels of ubiquinone-8 contribute to the distinctive but variable yellow color of different types of Bdellovibrio cells. Steady-state ubiquinone-8 concentrations did not differ markedly between conventional predatory and host-independent B. bacteriovorus despite upregulation of a suite of ubiquinone-8 synthesis genes in host-independent cells. In contrast, in spatially organized B. bacteriovorus films, the yellow inner regions contain significantly higher ubiquinone-8 concentrations than the off-white outer regions. Correspondingly, RT-PCR analysis reveals that the inner region, previously shown to consist primarily of active predators, clearly expresses two ubiquinone biosynthesis genes, while the outer region, composed mainly of quiescent or stalled bdelloplasts, expresses those genes weakly or not at all. Moreover, B. bacteriovorus cells in the inner region of week-old interfacial films, which are phenotypically attack-phase, have much higher UQ8 levels than regular attack-phase bdellovibrios, most likely because their "trapped" state prevents a high expenditure of energy to power flagellar motion.