Formation of biofilms under phage predation: considerations concerning a biofilm increase

<div><p>Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (<i>Pseudomonas aeruginosa</i>, <i>Salmonella enterica</i> serotype Typhimurium, and <i>Staphylococcus aureus</i>) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of <i>P. aeruginosa</i>. For biofilms of <i>S. aureus</i> and <i>S. enterica</i> Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.</p> </div

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

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

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

Molecular structure of alkaloids used in this study.

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

Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active Cargo Delivery

Biohybrid cell-driven microsystems offer unparalleled possibilities for realization of soft microrobots at the micron scale. Here, we introduce a bacteria-driven microswimmer that combines the active locomotion and sensing capabilities of bacteria with the desirable encapsulation and viscoelastic properties of a soft double-micelle microemulsion for active transport and delivery of cargo (<i>e</i>.<i>g</i>., imaging agents, genes, and drugs) to living cells. Quasi-monodisperse double emulsions were synthesized with an aqueous core that encapsulated the fluorescence imaging agents, as a proof-of-concept cargo in this study, and an outer oil shell that was functionalized with streptavidin for specific and stable attachment of biotin-conjugated <i>Escherichia coli</i>. Motile bacteria effectively propelled the soft microswimmers across a Transwell membrane, actively delivering imaging agents (<i>i</i>.<i>e</i>., dyes) encapsulated inside of the micelles to a monolayer of cultured MCF7 breast cancer and J744A.1 macrophage cells, which enabled real-time, live-cell imaging of cell organelles, namely mitochondria, endoplasmic reticulum, and Golgi body. This <i>in vitro</i> model demonstrates the proof-of-concept feasibility of the proposed soft microswimmers and offers promise for potential biomedical applications in active and/or targeted transport and delivery of imaging agents, drugs, stem cells, siRNA, and therapeutic genes to live tissue in <i>in vitro</i> disease models (<i>e</i>.<i>g</i>., organ-on-a-chip devices) and stagnant or low-flow-velocity fluidic regions of the human body

Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active Cargo Delivery

Biohybrid cell-driven microsystems offer unparalleled possibilities for realization of soft microrobots at the micron scale. Here, we introduce a bacteria-driven microswimmer that combines the active locomotion and sensing capabilities of bacteria with the desirable encapsulation and viscoelastic properties of a soft double-micelle microemulsion for active transport and delivery of cargo (<i>e</i>.<i>g</i>., imaging agents, genes, and drugs) to living cells. Quasi-monodisperse double emulsions were synthesized with an aqueous core that encapsulated the fluorescence imaging agents, as a proof-of-concept cargo in this study, and an outer oil shell that was functionalized with streptavidin for specific and stable attachment of biotin-conjugated <i>Escherichia coli</i>. Motile bacteria effectively propelled the soft microswimmers across a Transwell membrane, actively delivering imaging agents (<i>i</i>.<i>e</i>., dyes) encapsulated inside of the micelles to a monolayer of cultured MCF7 breast cancer and J744A.1 macrophage cells, which enabled real-time, live-cell imaging of cell organelles, namely mitochondria, endoplasmic reticulum, and Golgi body. This <i>in vitro</i> model demonstrates the proof-of-concept feasibility of the proposed soft microswimmers and offers promise for potential biomedical applications in active and/or targeted transport and delivery of imaging agents, drugs, stem cells, siRNA, and therapeutic genes to live tissue in <i>in vitro</i> disease models (<i>e</i>.<i>g</i>., organ-on-a-chip devices) and stagnant or low-flow-velocity fluidic regions of the human body