Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms.

Methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and PCR. However, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. We have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. The probability of plasmid segregational loss in planktonic and biofilm cultures of Pseudomonas putida carrying the TOL plasmid (pWWO::gfpmut3b) was determined directly in situ, in the absence of any applied selection pressure. Compared to suspended liquid culture, we report that the biofilm mode of growth enhances plasmid segregational loss. Results based on a biofilm-averaged analysis reveal that the probability of plasmid loss in biofilm cultures (0.016 ± 0.004) was significantly greater than that determined in planktonic cultures (0.0052 ± 0.0011). Non-invasive assessments showed that probabilities of plasmid segregational loss at different locations in a biofilm increased dramatically from 0.1% at the substratum surface to 8% at outside layers of biofilm. Results suggest that higher nutrient concentrations and subsequentially higher growth rates resulted in higher probability of plasmid segregational loss at the outer layers of the biofilm.

Artificial opsonin enhances bacterial phagocytosis, oxidative burst and chemokine production by human neutrophils.

Here, we describe the application of an 'artificial opsonin' to stimulate the innate immune response against Gram-positive bacteria. The artificial opsonin comprises a poly(L-lysine)-graft-poly(ethylene glycol) backbone displaying multiple copies of vancomycin and human IgG-Fc. The vancomycin targets bacteria by recognizing d-Ala-d-Ala-terminated peptides present in the bacterial cell wall. The human IgG-Fc antibody fragments serve as phagocyte recognition moieties that recognize the Fcγ cell surface receptors expressed by professional human phagocytes. Staphylococcus epidermidis RP62A, a biofilm-forming, methicillin-resistant strain, was utilized to investigate the effects of opsonization on phagocytosis, oxidative burst and IL-8 chemokine production by human neutrophils. Results show that opsonization of S. epidermidis RP62A with the artificial opsonin resulted in an ∼2-fold increase in neutrophil phagocytosis. Analysis of the cell supernatant found a 2- to 3-fold increase in neutrophil IL-8 secretion. The neutrophil oxidative burst was investigated using the oxidation-sensitive fluorophore dihydrorhodamine-123. Bacterial opsonization resulted in a 20% increase in fluorescence intensity, indicating a significant increase in the production of reactive oxygen species by the neutrophils. These studies suggest that artificial opsonins may be a novel immunostimulation therapeutic strategy to control infections caused by Gram-positive bacteria, particularly those that are known to be immune evasive and/or antibiotic resistant.

Multivalent artificial opsonin for the recognition and phagocytosis of Gram-positive bacteria by human phagocytes.

Hospital-acquired infections (HAIs) remain a leading cause of death in the United States. Unfortunately, treatment of HAIs is complicated by the emergence of antibiotic-resistant bacterial strains. In an effort to enhance the body's natural immune response to infection, we have developed an artificial opsonin to promote the recognition, phagocytosis, and destruction of pathogenic bacteria by human phagocytes. The artificial opsonin is constructed from multivalent conjugates of poly(L-lysine)-graft-poly(ethylene glycol) with vancomycin and human IgG-Fc. Our approach utilizes vancomycin's inherent ability to bind to D-Ala-D-Ala terminated peptides present in the cell wall of Gram-positive bacteria. Here, we show that conjugation of vancomycin to PLL-g-PEG prevents its action as an antibiotic and allows vancomycin to function solely as a recognition molecule. Human IgG-Fc antibody fragment serves as a phagocyte recognition molecule and is recognized by the Fcγ cell surface receptors expressed on professional human phagocytes. Using flow cytometry, we found that a polysaccharide-encapsulated, methicillin-resistant strain of Staphylococcus epidermidis is efficiently recognized by the artificial opsonin (nearly 100% of cells were opsonized) and that opsonin binding is specific since it can be inhibited by the soluble cell wall peptide analog acetyl-Lys-D-Ala-D-Ala. Opsonization of S. epidermidis resulted in an approximate 2-fold increase in phagocytosis by a human neutrophil cell line. Notably, Enterococcus faecalis VanB, a bacterial strain with inducible vancomycin resistance, was used to show that the artificial opsonin does not unintentionally induce antibiotic resistance mechanisms.