Research ArticleAPPLIED SCIENCES AND ENGINEERING

Self-targeting, zwitterionic micellar dispersants enhance antibiotic killing of infectious biofilms—An intravital imaging study in mice

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Science Advances  14 Aug 2020:
Vol. 6, no. 33, eabb1112
DOI: 10.1126/sciadv.abb1112
  • Fig. 1 Characterization of ZW-MSPMs and SSPMs prepared.

    (A) 1H NMR spectra of the PQAE conversion in trifluoroacetic acid. The peak shift from A [3.92 parts per million (ppm)] to B (4.54 ppm) indicates the conversion of the ZW structure to a lactone ring structure. (B) Hydrodynamic diameter distributions of SSPMs and ZW-MSPMs at pH 7.4 in phosphate-buffered saline (PBS; 10 mM potassium phosphate and 150 mM NaCl). (C) Transmission electron microscopy (TEM) micrographs of SSPMs and ZW-MSPMs at pH 7.4. (D) Zeta potentials of ZW-MSPMs and SSPMs in PBS as a function of time measured at pH 5.0 (37°C). (E) Zeta potentials of ZW-MSPMs and SSPMs as a function of pH measured in PBS (37°C), measured after 120-min exposure to each pH. All error bars denote SDs over three separately prepared batches of micelles.

  • Fig. 2 Self-targeting in S. aureus ATCC12600GFP biofilms of Nile red–loaded ZW-MSPMs in vitro and in vivo.

    (A) In vitro self-targeting of Nile red–loaded ZW-MSPMs into green fluorescent, 48-hour-old biofilms after 30 min of exposure to Nile red–loaded ZW-MSPMs suspended in PBS. (B) In vitro accumulation of Nile red–loaded ZW-MSPMs as a function of biofilm height. Accumulation was normalized with respect to the maximum intensity (pH 5.0). (C) Normalized green and red fluorescence as a function of biofilm height after in vitro accumulation of ZW-MSPMs at pH 5.0. Overlap of both curves indicates targeting of ZW-MSPMs to staphylococci. (D) A mouse with an abdominal imaging window (AIW) implanted in its flank underneath in which a green fluorescent staphylococcal biofilm was grown. (E) Custom-designed microscope stage, with the window frame fixed in a clamp to ensure proper intravital focusing over time. (F) Reconstructed three-dimensional (3D) intravital images of green fluorescent, 48-hour-old staphylococcal biofilms grown underneath the AIW, taken 30 min after injection of a Nile red–loaded SSPM suspension in PBS (pH 7.4). The bottom intravital image is the red fluorescence channel, taken 30 min after injection of a Nile red–loaded SSPMs suspension in mice without a staphylococcal biofilm grown underneath the window. (G) Same as (F), demonstrating in vivo accumulation of red fluorescent ZW-MSPMs in the 48-hour-old biofilm. (H) Red fluorescence intensity due to Nile red–loaded ZW-MSPMs accumulated in a 48-hour-old biofilm underneath the window as a function of time after tail injection of micelles. Intravital images were taken every 2 min. In three mice, without a biofilm grown underneath the window, no detectable red fluorescence due to micelle accumulation was observed. Red fluorescence intensity was normalized with respect to the maximum intensity after 28 min. Photo credit: (D and E) Shuang Tian, Nankai University and University Medical Center Groningen.

  • Fig. 3 Dispersal of 48-hour-old S. aureus ATCC12600GFP biofilms upon 120 min of exposure to ZW-MSPMs or SSPMs in PBS (pH 5.0).

    (A) CV-stainable bacterial biomass relative to PBS (pH 5.0), derived from optical density at 595 nm (OD595nm) after CV staining of in vitro–grown S. aureus biofilms as a function of micelle concentration in suspension. (B) Confocal laser scanning microscopy (CLSM) micrographs of staphylococcal biofilms exposed to PBS or suspensions (200 μg ml−1) of SSPMs and ZW-MSPMs. (C) Thickness of staphylococcal biofilms after exposure to PBS or suspensions of SSPMs or ZW-MSPMs. (D) Example of biomass distribution after exposure to PBS, or suspensions (200 μg ml−1) of SSPMs and ZW-MSPMs, across the height of the staphylococcal biofilms. Bacterial biomass was derived from green fluorescent pixels, while EPS mass was derived from red fluorescent pixels. Maximal green and red fluorescence intensities after exposure were set to 100%. (E) Biomass relative to PBS of bacteria and EPS, derived from COMSTAT analysis of CLSM images of staphylococcal biofilms exposed to micellar suspensions [micelles (200 μg ml−1)]. All error bars denote SDs over three experiments with separately prepared micelle batches and differently grown staphylococcal cultures. ** denotes statistically significant differences at P < 0.01 (Student’s t test).

  • Fig. 4 Interactions of SSPMs or ZW-MSPMs suspended in PBS (pH 5.0) with two major staphylococcal EPS components and effects on a 48-hour-old biofilm (scanning electron microscopy).

    (A) Effects of exposure of DNA for 120 min to PBS or suspensions (200 μg ml−1) of SSPMs or ZW-MSPMs. DNA interaction was assessed by an ethidium bromide (EB) displacement assay, with 100% coinciding with full displacement of EB. (B) Same as (A), for interaction with proteinaceous amyloidal fibrils. Amyloid interaction was assessed by a thioflavin T (ThT) fluorescence assay, with 100% coinciding with full disruption of amyloid fibrils. Error bars denote SDs over three experiments with separately prepared micelle batches. *** and **** indicate statistically significant differences at P < 0.001 and P < 0.0001, respectively (Student’s t test). (C) Scanning electron microscopy (SEM) images of 48-hour-old S. aureus ATCC12600GFP biofilms after 120 min of exposure to PBS or suspensions (200 μg ml−1) of SSPMs and ZW-MSPMs, demonstrating EPS patches in biofilms exposed to PBS and SSPMs but not after exposure to ZW-MSPMs.

  • Fig. 5 Antibiotic effects on 48-hour-old S. aureus ATCC12600GFP biofilm remaining after dispersal by exposure to ZW-MSPMs or SSPMs suspended in PBS (pH 5.0).

    (A) CLSM images of the penetration of red fluorescent ciprofloxacin (CIP) in in vitro–grown S. aureus biofilms after 120 min of exposure to PBS or suspensions (200 μg ml−1) of SSPMs or ZW-MSPMs. (B) Red fluorescence due to ciprofloxacin penetration, as a function of height in staphylococcal biofilms, derived from CLSM images (A). (C) Viability of staphylococci in a biofilm mode after pre-exposure to TSB or suspensions (200 μg ml−1) of SSPMs and ZW-MSPMs for 120 min and subsequent exposure to various concentrations of ciprofloxacin in TSB for 5 hours. Staphylococcal viability was assessed from the green fluorescence of the biofilms. Green fluorescence of biofilms before ciprofloxacin exposure was set to 100%. (D) Number of S. aureus colonies formed per square centimeter well surface in a biofilm mode, pre-exposed to tryptone soya broth (TSB) or suspensions (200 μg ml−1) of SSPMs and ZW-MSPMs for 120 min and subsequently exposed to ciprofloxacin (40 μg ml−1) for 5 hours. Error bars denote SDs over three experiments with separately prepared micelle batches and differently grown staphylococcal cultures. **** indicates statistically significant differences at P < 0.0001 (Student’s t test).

  • Fig. 6 In vivo efficacy of antibiotic treatment of an infectious S. aureus ATCC12600GFP, pretreated with ZW-MSPMs or SSPMs.

    (A) Experimental scheme for experiments using intravital imaging in mice equipped with an AIW. Consecutive tail injections of micelles and ciprofloxacin were performed daily, starting at day 0, i.e., 48 hours after initiating growth of an infectious biofilm (day −2). Injection with PBS, SSPMs, or ZW-MSPMs was immediately followed by injection of ciprofloxacin. (B) Reconstructed 3D intravital images of green fluorescent S. aureus biofilms before treatment at day 0 and after tail injection of PBS or SSPMs and ZW-MSPMs suspended in PBS (pH 7.4; 200 μg ml−1), immediately followed by ciprofloxacin in PBS (1 mg ml−1). Images were taken at different days up to sacrifice at day 5. (C) Thickness of staphylococcal biofilms underneath the AIW as a function of time after initiating treatment, derived from COMSTAT analysis of intravital images. (D) Same as (C), now for biomass. (E) Number of S. aureus CFUs obtained from the AIW and infection site tissue excised after sacrifice at day 5. Error bars denote SDs over three mice. **** indicates statistically significant differences at P < 0.0001 (Student’s t test). ns, not significant.

Supplementary Materials

  • Supplementary Materials

    Self-targeting, zwitterionic micellar dispersants enhance antibiotic killing of infectious biofilms—An intravital imaging study in mice

    Shuang Tian, Linzhu Su, Yong Liu, Jingjing Cao, Guang Yang, Yijin Ren, Fan Huang, Jianfeng Liu, Yingli An, Henny C. van der Mei, Henk J. Busscher, Linqi Shi

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