This study describes Chromobacterium violaceum's use of extracellular membrane vesicles (MVs) to both solubilize and transport violacein to other microorganisms. Violacein is a hydrophobic bisindole with known antibiotic activities against other microorganisms. Characterization of the MVs found they carried more violacein than protein (1.37 AE 0.19-fold), suggesting they may act as a reservoir for this compound. However, MVs are not produced in response to violaceina ΔvioA isogenic mutant, which is incapable of making violacein, actually produced significantly more MVs (3.2-fold) than the wild-type strain. Although violacein is insoluble in water (Log P octanol: water = 3.34), 79.5% remained in the aqueous phase when it was present within the C. violaceum MVs, an increase in solubility of 1740-fold. Moreover, tests with a strain of Staphylococcus aureus showed MVassociated violacein is bactericidal, with 3.

Bdellovibrio bacteriovorus HD100 is a predatory bacterium which lives by invading the periplasm of Gram-negative bacteria and consuming them from within. Although B. bacteriovorus HD100 attacks only Gram-negative bacterial strains, our work here shows attack-phase predatory cells also benefit from interacting with Gram-positive biofilms. Using Staphylococcus aureus biofilms, we show this predator degrades the biofilm matrix, obtains nutrients and uses these to produce and secrete proteolytic enzymes to continue this process. When exposed to S. aureus biofilms, the transcriptome of B. bacteriovorus HD100 was analogous to that seen when present intraperiplasmically, suggesting it is responding similarly as when in a prey. Moreover, two of the induced proteases (Bd2269 and Bd2692) were purified and their activities against S. aureus biofilms verified. In addition, B. bacteriovorus HD100 gained several clear benefits from its interactions with S. aureus biofilms, including increased ATP pools and improved downstream predatory activities when provided prey.

Violacein is a bisindole antibiotic that is effective against Gram-positive bacteria while the bacterial predator, Bdellovibrio bacteriovorus HD100, predates on Gram-negative strains. In this study, we evaluated the use of both together against multidrug resistant pathogens. The two antibacterial agents did not antagonize the activity of the other. For example, treatment of Staphylococcus aureus with violacein reduced its viability by more than 2,000-fold with or without B. bacteriovorus addition. Likewise, predation of Acinetobacter baumannii reduced the viability of this pathogen by more than 13,000-fold, regardless if violacein was present or not. When used individually against mixed bacterial cultures containing both Gram-positive and Gram-negative strains, violacein and B. bacteriovorus HD100 were effective against only their respective strains. The combined application of both violacein and B. bacteriovorus HD100, however, reduced the total pathogen numbers by as much as 84,500-fold. Their combined effectiveness was also demonstrated using a 4-species culture containing S. aureus, A. baumannii, Bacillus cereus and Klebsiella pneumoniae. When used alone, violacein and bacterial predation reduced the total population by only 19% and 68%, respectively. In conjunction with each other, the pathogen viability was reduced by 2,965-fold (99.98%), illustrating the prospective use of these two antimicrobials together against mixed species populations.

We evaluated the bactericidal activity of Bdellovibrio bacteriovorus, strain HD100, within blood sera against bacterial strains commonly associated with bacteremic infections, including E. coli, Klebsiella pneumoniae and Salmonella enterica. Tests show that B. bacteriovorus HD100 is not susceptible to serum complement or its bactericidal activity. After a two hour exposure to human sera, the prey populations decreased 15- to 7,300-fold due to the serum complement activity while, in contrast, the B. bacteriovorus HD100 population showed a loss of only 33%. Dot blot analyses showed that this is not due to the absence of antibodies against this predator. Predation in human serum was inhibited, though, by both the osmolality and serum albumin. The activity of B. bacteriovorus HD100 showed a sharp transition between 200 and 250 mOsm/kg, and was progressively reduced as the osmolality increased. Serum albumin also acted to inhibit predation by binding to and coating the predatory cells. This was confirmed via dot blot analyses and confocal microscopy. The results from both the osmolality and serum albumin tests were incorporated into a numerical model describing bacterial predation of pathogens. In conclusion, both of these factors inhibit predation and, as such, they limit its effectiveness against pathogenic prey located within sera.

SUMMARY Streptococcus pneumoniae (Spn) alone and during co-infection with influenza A virus (IAV) can result in severe pneumonia with mortality. Pneumococcal surface protein A (PspA) is an established virulence factor required for Spn evasion of lactoferricin and C-reactive protein-activated complement-mediated killing. Herein, we show that PspA functions as an adhesin to dying host cells. We demonstrate that PspA binds to host-derived glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bound to outward-flipped phosphatidylserine residues on dying host cells. PspA-mediated adhesion was to apoptotic, pyroptotic, and necroptotic cells, but not healthy lung cells. Using isogenic mutants of Spn , we show that PspA-GAPDH-mediated binding to lung cells increases pneumococcal localization in the lower airway, and this is enhanced as a result of pneumolysin exposure or co-infection with IAV. PspA-mediated binding to GAPDH requires amino acids 230–281 in its α-helical domain with intratracheal inoculation of this PspA fragment alongside the bacteria reducing disease severity in an IAV/ Spn pneumonia model.

Predation of Chromobacterium piscinae by Bdellovibrio bacteriovorus HD100 was inhibited in dilute nutrient broth (DNB) but not in HEPES. Experiments showed that the effector responsible was present in the medium, as cell-free supernatants retained the ability to inhibit predation, and that the effector was not toxic to B. bacteriovorus. Violacein, a bisindole secondary metabolite produced by C. piscinae, was not responsible. Further characterization of C. piscinae found that this species produces sufficient concentrations of cyanide (202 µM) when grown in DNB to inhibit the predatory activity of B. bacteriovorus, but that in HEPES, the cyanide concentrations were negligible (19 µM). The antagonistic role of cyanide was further confirmed, as the addition of hydroxocobalamin, which chelates cyanide, allowed predation to proceed. The activity of cyanide against B. bacteriovorus was found to be twofold, depending on the life cycle stage of this predator. For the attack-phase predatory cells, cyanide caused the cells to lose motility and tumble, while for intraperiplasmic predators, development and lysis of the prey cell were halted. These findings suggest that cyanogenesis in nature may be employed by the bacterial strains that produce this compound to prevent and reduce their predation by B. bacteriovorus.

The prevalence of Staphylococcus aureus worldwide as a nosocomial infectious agent is recognized but the reason behind the spread of this bacterium has remained elusive. Here, we hypothesized that the communication of S. aureus might benefit from it blocking other bacteria from establishing themselves on the surface. This was found to be the case for several pathogens as the S. aureus supernatant curtailed their ability to form biofilms. Subsequent analyses using Acinetobacter baumannii as a model found this effect is primarily mediated by S. aureus’ extracellular vesicles (EVs), which bound to the polystyrene surface. We found the EV-treated surfaces were significantly more hydrophilic after EV treatment, a condition that made it difficult for A. baumannii to initially adhere to the polystyrene surface and reduced its resulting biofilm by up to 93%. Subsequent tests found this also extended to several other bacterial pathogens, with a 40-70% decrease in their biofilm mass. The S. aureus EVs and their activity still remained after the surface was washed with 10% bleach, while the use of ethylenediaminetetraacetic acid (EDTA) removed both the EVs from the surface and their activity.

This study assessed the dynamics of predation by Bdellovibrio bacteriovorus HD 100. Predation tests with two different bioluminescent strains of Escherichia coli, one expressing a heat-labile bacterial luciferase and the other a heat-stable form, showed near identical losses from both, indicating that protein expression and stability are not responsible for the "shutting-off" of the prey bioluminescence (BL). Furthermore, it was found that the loss in the prey BL was not proportional with the predator-to-prey ratio (PPR), with significantly greater losses seen as this value was increased. This suggests that other factors also play a role in lowering the prey BL. The loss in BL, however, was very consistent within nine independent experiments to the point that we were able to reliably estimate the predator numbers within only 1 h when present at a PPR of 6 or higher, Using a fluorescent prey, we found that premature lysis of the prey occurs at a significant level and was more prominent as the PPR ratio increased. Based upon the supernatant fluorescent signal, even a relatively low PPR of 10-20 led to approximately 5% of the prey population being prematurely lysed within 1 h, while a PPR of 90 led to nearly 15% lysis. Consequently, we developed a modified Lotka-Volterra predator-prey model that accounted for this lysis and is able to reliably estimate the prey and bdelloplast populations for a wide range of PPRs.