Ehud Banin

Multi-species biofilms: living with friendly neighbors

Our knowledge regarding the nature and development of microbial biofilms has grown significantly since the first report of these communities by Antonie van Leeuwenhoek in the late 1600s. Nevertheless, most biofilm studies examine mono-species cultures, whereas nearly all biofilm communities in nature comprise a variety of microorganisms. The species that constitute a mixed biofilm and the interactions between these microorganisms critically influence the development and shape of the community. In this review, we focus on interactions occurring within a multi-species biofilm and their effects on the nature of the mixed community. In general, interspecies interactions involve communication, typically via quorum sensing, and metabolic cooperation or competition. Interactions among species within a biofilm can be antagonistic, such as competition over nutrients and growth inhibition, or synergistic. The latter can result in the development of several beneficial phenotypes. These include the promotion of biofilm formation by co-aggregation, metabolic cooperation where one species utilizes a metabolite produced by a neighboring species, and increased resistance to antibiotics or host immune responses compared to the mono-species biofilms. These beneficial interactions in mixed biofilms have important environmental, industrial, and clinical implications. The latter, for example, impacts the course and treatment of biofilm-related infections, such as those manifested in the lungs of cystic fibrosis patients.

Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica

The aetiological agent of bleaching of the coral Oculina patagonica was characterized as a new Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data and phenotypic properties, including the cellular fatty acid profile. Based on its 16S rDNA and DNA-DNA hybridization, the new Vibrio species is closely related to Vibrio mediterranei. The name Vibrio shiloi sp. nov. is proposed for the new coral-bleaching species, the type strain being AK1T (= ATCC BAA-91T = DSM 13774T).

Understanding the Antibacterial Mechanism of CuO Nanoparticles: Revealing the Route of Induced Oxidative Stress

To date, there is still a lack of definite knowledge regarding the interaction of CuO nanoparticles with bacteria and the possible permeation of the nanoparticles into bacterial cells. This study was aimed at shedding light on the size-dependent (from the microscale down to the small nanoscale) antibacterial activity of CuO. The potent antibacterial activity of CuO nanoparticles was found to be due to ROS-generation by the nanoparticles attached to the bacterial cells, which in turn provoked an enhancement of the intracellular oxidative stress. This paradigm was confirmed by several assays such as lipid peroxidation and reporter strains of oxidative stress. Furthermore, electron microscopy indicated that the small nanoparticles of CuO penetrated the cells. Collectively, the results reported herein may reconcile conflicting concepts in the literature concerning the antibacterial mechanism of CuO nanoparticles, as well as highlight the potential for developing sustainable CuO nanoparticles-based devices for inhibiting bacterial infections.

The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent

The opportunistic pathogen Pseudomonas aeruginosa causes infections that are difficult to treat by antibiotic therapy. This bacterium can cause biofilm infections where it shows tolerance to antibiotics. Here we report the novel use of a metallo-complex, desferrioxamine-gallium (DFO-Ga) that targets P. aeruginosa iron metabolism. This complex kills free-living bacteria and blocks biofilm formation. A combination of DFO-Ga and the anti-Pseudomonas antibiotic gentamicin caused massive killing of P. aeruginosa cells in mature biofilms. In a P. aeruginosa rabbit corneal infection, topical administration of DFO-Ga together with gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of gentamicin alone. The use of DFO-Ga as a Trojan horse delivery system that interferes with iron metabolism shows promise as a treatment for P. aeruginosa infections.

Penetration of the Coral-Bleaching Bacterium Vibrio shiloi into Oculina patagonica

ABSTRACT Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a β-d-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29°C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloiantibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.

Influence of Quorum Sensing and Iron on Twitching Motility and Biofilm Formation in Pseudomonas aeruginosa

Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at FeCl3 concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of FeCl3 that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhlI, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhlI mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.

Antibiofilm activity of nanosized magnesium fluoride

The ability of bacteria to develop antibiotic resistance and colonize abiotic surfaces by forming biofilms is a major cause of medical implant-associated infections and results in prolonged hospitalization periods and patient mortality. This raises the urgent need to develop compounds that can inhibit bacterial colonization of surfaces. In this study, we present an unreported microwave-based synthesis of MgF(2) nanoparticles (Nps) using ionic liquid. We demonstrate the antimicrobial activity of these fluoride nanomaterials and their ability to restrict biofilm formation of common bacterial pathogens. Scanning and transmission electron microscopic techniques indicated that the MgF(2).Nps attach and penetrate into the cells. Flow cytometry analysis revealed that the Nps caused a disruption in the membrane potential. The MgF(2).Nps also induced membrane lipid peroxidation and once internalized can interact with chromosomal DNA. Based on these findings we further explored the possibility of using the MgF(2).Nps to coat surfaces and inhibit biofilm formation. A microwave synthesis and coating procedure was utilized to coat glass coupons. The MgF(2) coated surfaces effectively restricted biofilm formation of the tested bacteria. Taken together these results highlight the potential for developing MgF(2) nanoparticles in order to inhibit bacterial infections.

Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae.

ABSTRACT The coral-bleaching bacterium Vibrio shiloibiosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH4Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH4Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH4Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH3 into the cell. It is known that uptake of NH3 into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.

ZnO nanoparticle-coated surfaces inhibit bacterial biofilm formation and increase antibiotic susceptibility

Nanotechnology is providing new ways to manipulate the structure and chemistry of surfaces to inhibit bacterial colonization. In this study, we evaluated the ability of glass slides coated with zinc oxide (ZnO) nanoparticles to restrict the biofilm formation of common bacterial pathogens. The generation of hydroxyl radicals, originating from the coated surface, was found to play a key role in antibiofilm activity. Furthermore, we evaluated the ability of the nanoparticle coating to enhance the antibacterial activity of commonly-used antibiotics. The ZnO nanoparticles were synthesized and deposited on the surface of glass slides using a one-step ultrasound irradiation process. Several physico-chemical surface characterization methods were performed to prove the long-term stability and homogenity of the coated films. Collectively, our findings may open a new door for utilizing ZnO nanoparticle films as antibiofilm coating of surfaces, thus providing a versatile platform for a wide range of applications both in medical and industrial settings, all of which are prone to bacterial colonization.

Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.