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Novel antimicrobials or new treatment strategies are urgently needed to treat Pseudomonas aeruginosa (P. aeruginosa) related infections and especially to address the problem of antibiotic resistance. We propose a novel strategy that combines the human antimicrobial peptide (AMP) LL37 with different antibiotics to find synergistic AMP-antibiotic combinations against P. aeruginosa strains in vitro. Our results showed that LL37 exhibited synergistic inhibitory and bactericidal effects against P. aeruginosa strains PAO1 and PA103 when combined with the antibiotics vancomycin, azithromycin, polymyxin B, and colistin. In addition, LL37 caused strong outer membrane permeabilization, as demonstrated through measurement of an increased uptake of the fluorescent probe N-phenyl-1-naphthylamine. The membrane permeabilization effects appear to explain why it was easier to rescue the effectiveness of the antibiotic toward the bacteria because the outer membrane of P. aeruginosa exhibits barrier function for antibiotics. Furthermore, the change in the zeta potential was measured for P. aeruginosa strains with the addition of LL37. Zeta potentials for P. aeruginosa strains PAO1 and PA103 were -40.9 and -10.9 mV, respectively. With the addition of LL37, negative zeta potentials were gradually neutralized. We found that positively charged LL37 can interact with and neutralize the negatively charged bacterial outer membrane through electrostatic interactions, and the process of neutralization is believed to have contributed to the increase in outer membrane permeability. Finally, to further illustrate the relationship between outer membrane permeabilization and the uptake of antibiotics, we used LL37 to make the outer membrane of P. aeruginosa strains more permeable, and minimum inhibitory concentrations (MICs) for several antibiotics (colistin, gentamicin, polymyxin B, vancomycin, and azithromycin) were measured. The MICs decreased were twofold to fourfold, in general. For example, the MICs of azithromycin and vancomycin decreased more than fourfold when against P. aeruginosa strain PAO1, which were the greatest decrease of any of the antibiotics tested in this experiment. As for PA103, the MIC of polymyxin B2 decreased fourfold, which was the strongest decrease seen for any of the antibiotics tested in this experiment. The increased uptake of antibiotics not only demonstrates the barrier role of the outer membrane but also validates the mechanism of synergistic effects that we have proposed. These results indicate the great potential of an LL37-antibiotic combination strategy and provide possible explanations for the mechanisms behind this synergy.

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.

Atomic force microscopy (AFM) was used to quantify the adhesion forces between Pseudomonas aeruginosa PAO1 and AK1401, and a representative model protein, bovine serum albumin (BSA). The two bacteria strains differ in terms of the structure of their lipopolysaccharide (LPS) layers. While PAO1 is the wild-type expressing a complete LPS and two types of saccharide units in the O-antigen (A(+) B(+)), the mutant AK1401 expresses only a single unit of the A-band saccharide (A(+) B(-)). The mean adhesion force (F(adh)) between BSA and AK1401 was 1.12 nN, compared to 0.40 nN for F(adh) between BSA and PAO1. In order to better understand the fundamental forces that would control bacterial-protein interactions at equilibrium conditions, we calculated interfacial free energies using the van Oss-Chaudhury-Good (VCG) thermodynamic modeling approach. The hydrogen bond strength was also calculated using a Poisson statistical analysis. AK1401 has a higher ability to participate in hydrogen bonding with BSA than does PAO1, which may be because the short A-band and absence of B-band polymer allowed the core oligosaccharides and lipid A regions to be more exposed and to participate in hydrogen and chemical bonding. Interactions between PAO1 and BSA were weak due to the dominance of neutral and hydrophilic sugars of the A-band polymer. These results show that bacterial interactions with protein-coated surfaces will depend on the types of bonds that can form between bacterial surface macromolecules and the protein. We suggest that strategies to prevent bacterial colonization of biomaterials can focus on inhibiting these bonds.

Atomic force microscopy (AFM) was used to characterize interactions between natural organic matter (NOM), and glass or bacteria. Poly(methacrylic acid) (PMA), soil humic Acid (SHA), and Suwannee River humic Acid (SRHA), were adsorbed to silica AFM probes. Adhesion forces (Fadh) for the interaction of organic-probes and glass slides correlated with organic molecular weight (MW), but not with radius of the organic aggregate (R), charge density (Q), or zeta potential (zeta). Two Pseudomonas aeruginosa strains with different lipopolysaccharides (LPS) were chosen: PAO1 (A+B+), whose LPS have common antigen (A-band) + O-antigen (B-band); and mutant AK1401 (A+B-). Fadh between bacteria and organics correlated with organic MW, R, and Q, but not zeta. PAO1 had lower Fadh with silica than NOM, which was attributed to negative charges from the B-band polymers causing electrostatic repulsion. AK1401 adhered stronger to silica than to the organics, perhaps because the absence of the B-band exposed underlying positively charged proteins. DLVO calculations could not explain the differences in the two bacteria or predict qualitative or quantitative trends in interaction forces in these systems. Molecular-level information from AFM studies can bring us closer to understanding the complex nature of bacterial-NOM interactions.

The roles of lipopolysaccharides (LPS) and extracellular polymers (ECP) on the adhesion of Pseudomonas aeruginosa PAO1 (expresses the A-band and B-band of O antigen) and AK1401 (expresses the A-band but not the B-band) to silicon were investigated with atomic force microscopy (AFM) and related to biopolymer physical properties. Measurement of macroscopic properties showed that strain AK1401 is more negatively charged and slightly more hydrophobic than strain PAO1 is. Microscopic AFM investigations of individual bacteria showed differences in how the biopolymers interacted with silicon. PAO1 showed larger decay lengths in AFM approach cycles, suggesting that the longer polymers on PAO1 caused greater steric repulsion with the AFM tip. For both bacterial strains, the long-range interactions we observed (hundreds of nanometers) were inconsistent with the small sizes of LPS, suggesting that they were also influenced by ECP, especially polysaccharides. The AFM retraction profiles provide information on the adhesion strength of the biopolymers to silicon (F(adh)). For AK1401, the adhesion forces were only slightly lower (F(adh) = 0.51 nN compared to 0.56 nN for PAO1), but the adhesion events were concentrated over shorter distances. More than 90% of adhesion events for AK1401 were at distances of <600 nm, while >50% of adhesion events for PAO1 were at distances of >600 nm. The sizes of the observed molecules suggest that the adhesion of P. aeruginosa to silicon was controlled by ECP, in addition to LPS. Steric and electrostatic forces each contributed to the interfacial interactions between P. aeruginosa and the silicon surface.

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between C. parapsilosis and both unmodified silicone rubber and P. aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 +/- 0.25 nN in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 +/- 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that C. parapsilosis may adhere to both silicone rubber and P. aeruginosa biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.