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.