Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are predatory bacteria that naturally—and obligately—prey on other Gram-negative bacteria, and their use has been proposed as a potential new approach to control microbial infection. The ability of predatory bacteria to prey on Gram-negative human pathogens in vitro is well documented; however, the in vivo safety and efficacy of predatory bacteria have yet to be fully assessed. In this study, we examined whether predatory bacteria can reduce bacterial burden in the lungs in an in vivo mammalian system. Initial safety studies were performed by intranasal inoculation of rats with predatory bacteria. No adverse effects or lung pathology were observed in rats exposed to high concentrations of predatory bacteria at up to 10 days postinoculation. Enzyme-linked immunosorbent assay (ELISA) of the immune response revealed a slight increase in inflammatory cytokine levels at 1 h postinoculation that was not sustained by 48 h. Additionally, dissemination experiments showed that predators were efficiently cleared from the host by 10 days postinoculation. To measure the ability of predatory bacteria to reduce microbial burden in vivo, we introduced sublethal concentrations of Klebsiella pneumoniae into the lungs of rats via intranasal inoculation and followed with multiple doses of predatory bacteria over 24 h. Predatory bacteria were able to reduce K. pneumoniae bacterial burden, on average, by more than 3.0 log10 in the lungs of most rats as measured by CFU plating. The work presented here provides further support for the idea of developing predatory bacteria as a novel biocontrol agent.

Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negative bacteria, making predatory bacteria potential alternatives to antibiotics for treating multi-drug resistant infections. While the ability of predatory bacteria to control bacterial infections in vitro is well documented, the in vivo effect of predators on a living host has yet to be extensively examined. In this study, respiratory and intravenous inoculations were used to determine the effects of predatory bacteria in mice. We found no reduction in mouse viability after intranasal or intravenous inoculation of B. bacteriovorus 109J, HD100 or M. aeruginosavorus. Introducing predators into the respiratory tract of mice provoked a modest inflammatory response at 1 hour post-exposure, but was not sustained at 24 hours, as measured by RT-qPCR and ELISA. Intravenous injection caused an increase of IL-6 in the kidney and spleen, TNF in the liver and CXCL-1/KC in the blood at 3 hours post-exposure, returning to baseline levels by 18 hours. Histological analysis of tissues showed no pathological changes due to predatory bacteria. Furthermore, qPCR detected predators were cleared from the host quickly and efficiently. This work addresses some of the safety concerns regarding the potential use of predatory bacteria as a live antibiotic.Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are small, highly motile, uniflagellate Gram-negative bacteria that prey naturally on other Gram-negative bacteria 1,2 . Recently, predatory bacteria have been considered as potential alternatives to traditional antibiotics for treating multi-drug resistant (MDR) Gram-negative bacterial infections. B. bacteriovorus have a predatory lifestyle where they attach to and enter the prey periplasm, multiply by exhausting the nutrients, lyse the cell, and then continue to seek out more prey to invade 1,3,4 . Micavibrio spp., in contrast, attach to, grow and kill prey at the surface of the prey cell in a 'vampire'-like fashion 2,5,6 . Bdellovibrio-and-like organisms (BALOs) are a promising potential novel agent against bacterial pathogens and present several advantages when considering their use for controlling infection 7 . Previous studies have confirmed the ability of predatory bacteria to control a broad range of important human pathogens in vitro, including MDR bacteria 8 , grown both planktonically and in biofilms [9][10][11] . In addition,

Predatory bacteria are Gram-negative bacteria that prey on other Gram-negative bacteria and have been considered as potential therapeutic agents against multi-drug resistant pathogens. In vivo animal models have demonstrated that predatory bacteria are non-toxic and non-immunogenic in rodents. In order to consider the use of predatory bacteria as live antibiotics, it is important to investigate their effect on human cells. The aim of this study was to determine the effect of Bdellovibrio bacteriovorus strains 109J and HD100, and Micavibrio aeruginosavorus strain ARL-13 on cell viability and inflammatory responses of five human cell lines, representative of clinically relevant tissues. We found that the predators were not cytotoxic to any of the human cell lines tested. Microscopic imaging showed no signs of cell detachment, as compared to predator-free cells. In comparison to an E. coli control, exposure to higher concentrations of the predators did not trigger a significant elevation of pro-inflammatory cytokines in four of the five human cell lines tested. Our work underlines the non-pathogenic attributes of predatory bacteria on human cells and highlights their potential use as live antibiotics against human pathogens.

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative proteobacteria that are obligate predators of other Gram-negative bacteria and are considered potential alternatives to antibiotics. Most studies focusing on predatory bacteria have been performed in vitro, thus the effect of predatory bacteria on a live host, including the impact on the ecology of the native microbiota, has yet to be fully examined. In this study, intrarectal inoculations of Sprague-Dawley rats with predatory bacteria were performed. Additionally, feces were collected for seven days post-inoculation to determine the effect on gut bacterial diversity. Rat colonic tissue exhibited no abnormal histopathological effects due to predatory bacteria. A modest increase in pro-inflammatory cytokines was measured in the colons of rats inoculated with predatory bacteria by 24 and 48 hours, with all but IL-13 returning to baseline by seven days. V4 16S rRNA gene sequencing of fecal DNA demonstrated minimal shifts in taxonomic representation over the week due to predatory bacteria. Changes in bacterial populations due to exposure to B. bacteriovorus are predicted to contribute to health, however, an overgrowth of Prevotella was observed due to exposure to M. aeruginosavorus. This study further addresses safety concerns associated with the potential use of predatory bacteria to treat infections.

Background: Shellfish hypersensitivity is amongst the most common food allergies. The major shellfish allergen was identified as tropomyosin. Here, we investigated the immediate hypersensitivity responses, IgE and cell-mediated immune response in mice sensitized with recombinant shrimp tropomyosin. Methods: Shrimp tropomyosin was cloned and expressed as a His-tagged fusion recombinant protein in Escherichia coli. Three- to 4-week-old BALB/c mice were sensitized by intragastric administration of recombinant tropomyosin (0.1 mg) plus cholera toxin (10 µg) on days 0, 12, 19 and 26 and challenged on day 33. Mice fed with phosphate-buffered saline plus cholera toxin were included as controls. Animals were monitored for immediate hypersensitive responses and tropomyosin-specific IgE over time. In addition, shrimp tropomyosin-specific CD4+ T cells, interleukin-4 and interferon-γ levels were determined from in vitro splenocyte cultures. A passive cutaneous anaphylaxis assay was also conducted. Results: Mice fed with shrimp tropomyosin developed swelling of the snout, increased scratching behavior and shrimp tropomyosin-specific IgE. Sera from tropomyosin-sensitized mice elicited vascular leakage in naïve mice in the passive cutaneous anaphylaxis assay. Shrimp tropomyosin-specific CD4+ T cell proliferations and elevated interleukin-4 over interferon-γ levels were evident in splenocyte cultures of tropomyosin-fed mice upon tropomyosin stimulation. In contrast, shrimp tropomyosin-specific IgE, CD4+ T cells and hypersensitive responses were absent in the control mice. Conclusion: We have generated a BALB/c model of shrimp allergy. This model provides a useful tool for evaluating the immunopathogenic mechanisms involved in shellfish hypersensitivity.

The proteobacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are obligate predators of Gram-negative bacteria, and have been proposed to be used to treat multidrug-resistant bacterial infections. The ability of predatory bacteria to reduce bacterial burden in vivo within the lungs of rats has been demonstrated, but it was unknown if predatory bacteria can attenuate systemic bacterial burden administered intravenously. In this study, we first assessed the safety of intravenous inoculation of predatory bacteria in rats. No rat morbidity or adverse histopathology of various organs due to predatory bacteria administration was observed. An increase in proinflammatory cytokines (TNFα and KC/GRO) was observed at two hours post-inoculation; however, cytokines returned to baseline levels by 18 hours. Furthermore, bacterial dissemination analysis demonstrated that predatory bacteria were efficiently cleared from the host by 20 days post-injection. To determine whether predatory bacteria could reduce bacterial burden in vivo, Klebsiella pneumoniae was injected into the tail veins of rats and followed with multiple doses of predatory bacteria over 16 or 24 hours. Predatory bacteria were unable to significantly reduce K. pneumoniae burden in the blood or prevent dissemination to other organs. The results suggest that predatory bacteria may not be effective for treatment of acute blood infections.

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