Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant

BACKGROUND: Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison. RESULTS: The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB. CONCLUSION: The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms

Complete Killing of Agar Lawn Biofilms by Systematic Spacing of Antibiotic-Loaded Calcium Sulfate Beads

Background: Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are the major causative agents of acute and chronic infections. Antibiotic-loaded calcium sulfate beads (ALCSB) are used in the management of musculoskeletal infections such as periprosthetic joint infections (PJI). Methods: To determine whether the number and spatial distribution of ALCSB are important factors to totally eradicate biofilms, ALCSBs containing vancomycin and tobramycin were placed on 24 h agar lawn biofilms as a single bead in the center, or as 16 beads placed as four clusters of four, a ring around the edge and as a group in the center or 19 beads evenly across the plate. Bioluminescence was used to assess spatial metabolic activity in real time. Replica plating was used to assess viability. Results: For both strains antibiotics released from the beads completely killed biofilm bacteria in a zone immediately adjacent to each bead. However, for PA extended incubation revealed the emergence of resistant colony phenotypes between the zone of eradication and the background lawn. The rate of biofilm clearing was greater when the beads were distributed evenly over the plate. Conclusions: Both number and distribution pattern of ALCSB are important to ensure adequate coverage of antibiotics required to eradicate biofilms

Effect of Tannic and Gallic Acids Alone or in Combination with Carbenicillin or Tetracycline on Chromobacterium violaceum CV026 Growth, Motility, and Biofilm Formation

Chromobacterium violaceum is an opportunistic pathogen that causes infections that are difficult to treat. The goal of this research was to evaluate the effect of selected tannins (tannic acid (TA) and gallic acid (GA)) on bacterial growth, motility, antibiotic (carbenicillin, tetracycline) susceptibility, and biofilm formation. Both tannins, particularly TA, impaired bacterial growth levels and swimming motilities at sub-minimum inhibitory concentrations (sub-MICs). In combination with tannins, antibiotics showed increased MICs, suggesting that tannins interfered with antibacterial activity. Sub-MICs of tetracycline or TA alone enhanced biofilm formation of C. violaceum; however, in combination, these compounds inhibited biofilm formation. In contrast, carbenicillin at sub-MICs was effective in inhibiting C. violaceum biofilm formation; however, in combination with lower concentrations of TA or GA, biofilms were enhanced. These results provide insights into the effects of tannins on C. violaceum growth and their varying interaction with antibiotics used to target C. violaceum infections

Penetration of antibiotics ciprofloxacin (CIP) and azithromycin (ATH) through pre-formed <i>E. coli</i> CFT073 biofilms in presence and absence of piperine (PIP) and reserpine (RES).

<p>* Indicates statistically different values (p<0.05) when compared with antibiotic alone.</p><p>Penetration of antibiotics ciprofloxacin (CIP) and azithromycin (ATH) through pre-formed <i>E. coli</i> CFT073 biofilms in presence and absence of piperine (PIP) and reserpine (RES).</p

Effects of PIP and RES on antibiotic penetration through biofilms.

<p>(A) Confocal image of 48 h <i>E. coli</i> CFT073 biofilm formed on polycarbonate membranes, 3-dimensional reconstruction. Cells were stained with the fluorescent SYTO9 dye and are shown in green. (B) Representative images of zones of inhibition due to different treatments.</p

Molecular structure of alkaloids used in this study.

<p>(A) piperine, molecular weight: 285.34 and (B) reserpine, molecular weight: 608.68.</p

Expression of genes responsible for motility and biofilm formation in <i>E. coli</i> CFT073 wild type after 48 h of growth in presence and absence of PIP and RES.

<p>Relative mRNA quantities were normalized to that of a housekeeping gene, <i>gap</i>A. Results represent mean fold change values ± SD (in parentheses) for three independent experiments with respect to control. Values in bold font indicate statistically significant differences in mRNA relative value with respect to the control (<i>p</i><0.05).</p><p>Expression of genes responsible for motility and biofilm formation in <i>E. coli</i> CFT073 wild type after 48 h of growth in presence and absence of PIP and RES.</p

Effect of PIP and RES on the efficacy of antibiotics (A) ciprofloxacin (CIP 5 µg/mL) and (B) azithromycin (ATH 15 µg/mL) towards fully developed <i>E. coli</i> CFT073 biofilms.

<p>The biofilms were allowed to form for 48 h and were subsequently incubated with the alkaloids for 24 h. Control indicates biofilms not treated with antibiotic or alkaloids. All biofilm values (OD₅₇₀) are normalized with growth (OD₆₀₀). Values for OD₅₇₀ are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112093#pone.0112093.s006" target="_blank">Figure S6</a>. Abbreviations: PIP 0.5 or RES 0.5, piperine or reserpine at 0.5 µg/mL (e.g., PIP 0.5 indicates piperine at 0.5 µg/mL). * indicates statistically significant (<i>p</i><0.05) decrease in biofilm level for alkaloid + antibiotic treatment compared to the respective antibiotic treatment (CIP 5 or ATH 15).</p

Effect of PIP and RES on 48 h biofilm levels of <i>E. coli</i> CFT073 (white bars) and the flagellar mutant <i>E. coli</i> Δ<i>fli</i>C (black bars) in (A) 96 well plates and (B) polystyrene tubes.

<p>The biofilm values (OD₅₇₀) were normalized with respect to growth (OD₆₀₀). Abbreviations: PIP 0.5 or RES 0.5, piperine or reserpine at 0.5 µg/mL (<i>e.g.</i>, PIP 0.5 indicates piperine at 0.5 µg/mL). Values shown denote the mean + SD for three experiments. * and ** indicate statistically significant differences with respect to the control with values <i>p</i><0.05 and <i>p</i><0.01, respectively.</p

Effects of PIP and RES on <i>E. coli</i> CFT073 motility and flagellin expression.

<p>(A) swimming and (B) swarming motility of <i>E. coli</i> CFT073, (C) expression of flagellin in <i>E. coli</i> P<i>fli</i>C-lux in presence and absence of PIP and RES. * indicates statistically significant difference in values (<i>p</i><0.05) with respect to the control.</p