Studies on the Predatory Bacterium Bdellovibrio bacteriovorus and Its Impact on Bacterial Biofilms

Abstract

Recently, due to the rapid spread of the multi-drug resistant (MDR) bacterial infections and the significantly decreased rate of discovering new antibiotics, besides also the increased regulations upon releasing chemical anti-bacterial agents in the environment, the sights are directed towards finding novel tools for fighting those harmful and pathogenic bacteria. Bdellovibrio bacteriovorus is a predatory bacterium which lives through attacking Gram-negative bacterial strains, including pathogens such as Salmonella enterica and Acinetobacter baumannii. Owing to this character, B. bacteriovorus was proposed as a promising bio-control agent. In the current study, we evaluated the potential of applying B. bacteriovorus as a tool for eradicating harmful and pathogenic bacteria as well as their biofilms. Initially, we started to investigate the ability of B. bacteriovorus to predate upon planktonic Gram-negative prey in liquid cultures and to degrade its DNA. For this, we used a fluorescent strain of Escherichia coli. The results found that this predator can rapidly reduce recombinant bacterial populations within aqueous environments by more than 7-log within 24 hours. Using RT-qPCR, it was demonstrated that the plasmid copy number within the culture was reduced by approximately 700-fold. Subsequently, to understand deeper about this predator and its predaton cycle, a genome-wide microarray analysis was performed to compare the gene expression profile during the attack phase with that of the intraperiplasmic growth phase at 15, 30, 60, 120, and 180 min post-predation. This gave us a clearer picture for B. bacteriovorus transcriptome and the differential gene expression during different stages of B. bacteriovorus life. In subsequent experiments, we evaluated the effects of two different bacterial signaling molecules; indole and DSF on B. bacteriovorus predation and behavior. DSF was found to delay B. bacteriovorus predation but only at high concentrations. In contrast, Indole at physiological concentrations significantly delayed predation on E. coli and S. enterica and completely inhibited predation when present at 2 mM concentration. Subsequent microarray and RT-qPCR analyses showed that indole has wide spectrum effects on B. bacteriovorus transcriptome especially the down-regulation of flagellar and ribosomal genes. After this initial characterization of the predation in liquid cultures, we moved towards predating the Gram-negative bacteria in biofilm status. B. bacteriovorus could effectively mitigate the biofilms made by E. coli, S. enterica, and A. baumannii in 96-well plates as well as on silica chips. To move closer towards the medical application of B. bacteriovorus as an antibacterial agent, we next evaluated its ability to predate pathogenic communities on epithelial cell layers in tissue culture plates. For this, we developed a new technique depends on the aqueous two phase system (ATPS) micro-patterning. This technique enabled us to form physically separated bacterial communities on human epithelial cell sheets. Consequently, we could simultaneously test the localized effects of three different bacterial species; E. coli, Shigella boydii, and Pseudomonas sp. on an epithelial cell layer. In subsequent experiments, we showed the ability of B. bacteriovorus to remove these bacterial communities from the bacteria-patterned epithelial cell layers. Importantly, predation was critical for protecting the underlying epithelial cells from the damaging effect of P. sp. As one sound limitation for the wide spread application of this predator is its inability to attack Gram-positive bacterial pathogens such as Staphylococcus aureus and Staphylococcus epidermidis which both are major causes of the biofilm associated nosocomial infections, we were curious how we can overcome this limitation. In our studies with B. bacteriovorus, we found that adding the culture supernatant of the host independent mutant (HIB) at concentration of 10% (v/v) can effectively prevent and remove established S. aureus and S. epidermidis biofilms. This effect was found to come from the hydrolytic enzymes present in the supernatant especially the serine proteases. Furthermore, we expanded upon this and further demonstrated that the wild-type host dependent B. bacteriovorus (HDB) itself is capable of degrading S. aureus and S. epidermidis biofilms as well as other Gram-positive biofilms from different genera. Again, that was also mainly due to the serine proteases present in the supernatant. To confirm this biofilm removing activity of B. bacteriovorus secreted proteases, several serine proteases were cloned from B. bacteriovorus. These enzymes were then expressed, purified and their activities against S. aureus biofilms were evaluated. Interestingly, the supernatant and its proteases were not effective against any of the Gram-negative biofilms tested. As such, we demonstrated the hitherto unknown capability of B. bacteriovorus to mitigate Gram-positive but not Gram-negative biofilms through its secreted proteases, besides doing the reverse through predation. Therefore, this predator is a promising tool for removing multi-species biofilms of both Gram-positive and Gram-negative strains. This was proven through using a model mixed biofilm made by S. aureus and S. enterica. As such, this finding furthers the potential application of B. bacteriovorus as an antibacterial agent and in the treatment of diseases. In conclusion, the results discussed here shed more light on different aspects of B. bacteriovorus life cycle and its interactions with other bacteria which will help to understand more about this unique predatory bacterium. In addition, it also emphasized the potential of applying B. bacteriovorus for eradicating pathogenic and harmful bacteria together with their biofilms.