Authors: Daniel E. Kadouri and Nancy D. Connell (Rutgers University, NJ, USA)
We might have a problem. Our co-evolution with bacteria could be defined as a never-ending arms race between microbial virulence factors and our own immune system. Since the introduction of antibiotics some 70 years ago, the field initially tilted to our advantage. However, our overuse and misuse of antibiotics has placed selective pressure on bacteria to develop resistance to our miracle drugs and infectious bacterial disease has yet again become a serious global public health threat .
Among the more threatening bacteria in need of control are the Gram-negative bacteria, with their ability to exchange genetic material, including genes coding for drug resistance, and their double cell membrane that makes the delivery of new antibiotics into the cell extremely difficult. Even more challenging, the reluctance of the pharmaceutical industry to invest in antibiotic discovery has resulted in a current lapse in the antibiotic development pipeline.
This perfect storm of increased antimicrobial drug resistance and lack of new antibiotics in development has pushed researchers to try to find alternatives to antibiotics and to re-examine technologies that were set aside in an era where small compound antibiotics ruled supreme. Among these are the antimicrobials such as antimicrobial peptides, bacteriocins, phages and the predatory bacteria.
Predatory bacteria; who are we? In general, predatory bacteria are prokaryotic microorganisms that are obligatory parasitic predators that feed on other bacteria. Predatory bacteria can be found anywhere in nature. The first ‘true’ predatory bacterium, Bdellovibrio bacteriovorus, was isolated from soil in 1963 ; since then, predatory bacteria have been isolated from fresh and brackish water, sewage, water reservoirs and seawater. Predatory bacteria have also been isolated from natural surface-attached microbial communities known as biofilms.
In addition to their presence in abiotic environmental habitats, new data suggest that predatory bacteria might also be a permanent and/or transient component of animal and human microbiota. In 2001 Schwudke et al. utilized 16S rRNA to detect predatory bacteria in samples isolated from chickens, horses and humans . In a 2013 study, B. bacteriovorus was found in intestinal and fecal specimens of patients . The same study also reported higher abundance of the predators in healthy patients as compared to those with chronic intestinal diseases, suggesting that they may contribute to health by serving as an ecological balancer of normal gut flora.
The presence of predatory bacteria in human specimens was recently validated in a study in which next-generation sequencing was used to monitor the lung microbiota composition of cystic fibrosis patients, where predatory bacteria were detected . With the rapid advances in DNA analysis and sequencing, and the ability to detect smaller microbial populations within complex microbial samples, it is tempting to speculate that our understanding of the role, dynamics and diversity of predatory bacteria as a part of the microbiota will increase in the near future.
The most studied predatory bacteria are those from the genus Bdellovibrio. Bdellovibrio sp are small, highly-motile, Gram-negative delta-proteobacteria that prey on other Gram-negative bacteria . B. bacteriovorus, one member which is well studied for its antimicrobial attributes, is characterized by a non-replicating attack phase in which it swims randomly in its environment, collides with its prey and burrows through the bacterial outer membrane. At this point, the predator will switch to its reproductive phase within the prey’s periplasm, elongating and dividing into several daughter cells as it grows, ending in lysis of the host bacterium to start a new round of predation. Other members of the genus, such as B. exovorus, does not invade the prey but will feed as it leeches nutrients from an external attachment site .
Like B. exovorus, Micavibrio aeruginosavorus also externally attaches to its prey as it feeds . This alpha-proteobacterium was also examined for its potential to be used as a live antibiotic. Although Bdellovibrio sp. and Micavibrio sp. are the most studied, they represent only a small subset of predatory bacteria that exist in nature, each exhibiting a unique host range and predatory lifestyle that might be exploited to serve as a therapeutic.
Predatory bacteria; we are really good at killing bacteria. Predatory bacteria are very effective in reducing prey microbial population in vivo. The host range specificity differs from predator to predator. B. bacteriovorus, for example, demonstrates an extremely broad range of host specificity, attacking many human Gram-negative pathogens, including bacteria from the genera Acinetobacter, Bordetella, Burkholderia, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonella, Shigella, Vibrio and Yersinia, among others [8,9]. Interestingly, the extent of predation on a specific prey varies from one strain to another within the same species. Other predatory bacteria, such as M. aeruginosavorus and B. exovorus, exhibit a more limited prey range [6,8].
Resistance is futile. While the mechanisms governing prey specificity are not known, the development of predation resistance in a normally susceptible bacterial species has not yet been observed. Several unsuccessful mutant screens were conducted by our group and by others, searching for transposon mutants that render a resistant prey sensitive or a sensitive prey resistant. Furthermore, we have been unable to enrich for genetically stable predation-resistant isolates following sequential predation, as is found with phage treatment. The inability to generate genetically stable predation-resistant mutants could be the result of the numerous mechanisms – some of which might represent essential functions that the predator utilizes during predation. Alternatively, the predator may quickly counter-adapt and evolve a counter-resistance mechanism, under pressure from its obligate parasitic lifestyle.
Since predation appears not to involve any of the metabolic pathways or cell structures that alter the susceptibility of pathogens to antibiotics, the specific antibiotic-resistant characteristics of prey do not impact predation. Studies aimed at evaluating the ability of the predators to prey upon multidrug-resistant bacteria reported that the predatory bacteria B. bacteriovorus and M. aeruginosavorus are able to predate several clinical samples of Gram-negative isolates which harbor a variety of potent ß-lactamases, as well as fluoroquinolone-resistant and mcr-1-positive colistin-resistant bacteria [10–12]. Since predation susceptibility does not change with the acquisition of antibiotic resistance, it could be argued that predatory bacteria may be used not only against existing drug-resistant pathogens, but also against pathogens with any new resistance mechanisms that might emerge in the future.
Predatory bacteria were also demonstrated to be very effective against biofilms [13,14]. Although biofilms provide bacteria with enhanced tolerance to antibiotics and a safe haven from many biological predators, such as protozoa and phage, they provide little protection against predatory bacteria, which easily penetrate the protecting biofilm structure. Moreover, with the close dense prey cell population, biofilms provide ‘easy hunting’, allowing the predators to move from one cell to another and proliferate as they consume the biofilm from within [13,14]. Finally, the low metabolic activity exhibited within biofilms, which also contributes to the antibiotic tolerance of the biofilm, poses no problem to the predators: they are not influenced by metabolic activity of their prey . Thus, predatory bacteria could be a suitable alternative to control biotic or abiotic biofilms by themselves or in concert with other antimicrobials.
Predatory bacteria; are we safe? In order to be considered as potential therapeutics, predatory bacteria must be safe for humans and animals. In vivo studies of various human cell lines challenged with high doses of predatory bacteria demonstrated no negative effect to the cells, with no signs of cell death, cell detachment, cell migration or inflammation as measured by pro-inflammatory cytokines production (see recent review by Negus et al. in ). In addition, predators did not inhibit wound healing. This observation also was confirmed in vivo model in which wounded rabbit corneas healed and closed normally following exposure to predatory bacteria . Recent work in which mice and rats were exposed to high doses of predatory bacteria via intranasal, intravenous and gastrointestinal tract all demonstrated no signs of animal discomfort, infection or increase in morbidity ; rabbit eyes were also not harmed by exposure to predators . Histopathological examination of tissues also showed no abnormal findings or signs of inflammation.
The inability of the predators to elicit an aggressive immune response was also confirmed by measuring cytokine production using ELISA and qPCR, which might be explained by the predators’ unique lipopolysaccharide structure (see review ). These studies also demonstrated that although the predators disseminate throughout the organs after inoculation, the bacteria are cleared from the host within 72 hours with no negative outcome to the animals. Finally, minimal population shifts were measured in the gut bacterial microbiota during 1 week following intracolonic inoculations of predators ; this phenomenon is a major advantage over current antibiotic treatment that frequently causes dramatic off-target dysbiosis of the gut bacterial microbiota population. These recent animal safety studies are in agreement to early studies that also reported no negative effects on animals following inoculation .
But do we work in vivo? Several studies have been conducted to measure the ability of predatory bacteria to control infection in vivo. In one study, B. bacteriovorus was orally administered to young chicks that were pre-dosed with a gut-colonizing Salmonella enterica. It was discovered that the predators were able to reduce significantly Salmonella numbers in bird gut cecal contents and limit abnormal cecal morphology, indicating reduced cecal inflammation . In a study conducted by our team, infectious K. pneumoniae bacteria were introduced intranasally into the lungs of rats, and predatory bacteria were administered at several intervals over 24 hours. Predatory bacteria administration was able to attenuate K. pneumoniae burden by over 3-log in the lungs of rats, as measured by CFU plating .
At this point, additional studies are needed to determine if the reduction in pathogenic prey is a result of active predation or a combined effect of the predators with the host innate immune system. This interpretation was suggested in a study in which the ability of B. bacteriovorus to control Shigella flexneri in a zebrafish infection model was shown to be supported by macrophage and neutrophil activity at the site of infection . In another study, predatory bacteria were not able to reduce the burden of K. pneumoniae administered intravenously. Thus, it might be argued that localized use of the predators into a confined space might be more effective in controlling infection than systemic application.
So what’s next? Additional studies need to be conducted to fully understand the potential of predatory bacteria to be administered as a live therapeutic to prevent and control infection. Yet the proof of concept studies described here, regarding the safety and efficacy of the predators in mammalian infection, are promising. The future development and use of predatory bacteria to prevent, inhibit and control infection will have to take place in a regulatory environment in which we are open to new non-chemical based therapeutics; we must recognize that in the very near future, treating infection, much like cancer treatment, can no longer be performed solely by a ‘magic-bullet’ – a one drug to treat all approach – but needs to involve several therapeutics used together to assault the infection and limit the pathogens’ ability to adapt and resist. We propose, for example, the use of predatory bacteria to compromise biofilm integrity followed by the use of a chemical-based drug or phage. With humanity’s possible return to a pre-antibiotic era, the time has come to think outside the box: with 3.5 billion years of microbial evolution, ‘the answer is out there’ and it might just be a predatory bacteria.
Conflict of interest
The authors have no actual or potential conflict of interest to disclose.
Research was sponsored by the U.S. Army Research Office and the Defense Advanced Research Projects Agency grant to D. E. Kadouri and N. D. Connell, under Cooperative Agreement Number W911NF-15-2-0036. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, DARPA, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation hereon.
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Daniel E. Kadouri 1 and Nancy D. Connell 2
1 Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ 07103, USA 2 Division of Infectious Disease, Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ 07103, USA