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Susceptibility

Mycobacterium abscessus Smooth and Rough Morphotypes Form Antimicrobial-Tolerant Biofilm Phenotypes but Are Killed by Acetic Acid

Gillian Clary, Smitha J. Sasindran, Nathan Nesbitt, Laurel Mason, Sara Cole, Abul Azad, Karen McCoy, Larry S. Schlesinger, Luanne Hall-Stoodley
Gillian Clary
aDepartment of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, Ohio, USA
bThe Ohio State University College of Medicine, Columbus, Ohio, USA
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Smitha J. Sasindran
aDepartment of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, Ohio, USA
bThe Ohio State University College of Medicine, Columbus, Ohio, USA
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Nathan Nesbitt
bThe Ohio State University College of Medicine, Columbus, Ohio, USA
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Laurel Mason
cDepartment of Microbiology, Ohio State University, Columbus, Ohio, USA
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Sara Cole
dOSU Campus Microscopy and Imaging Facility, Ohio State University, Columbus, Ohio, USA
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Abul Azad
aDepartment of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, Ohio, USA
bThe Ohio State University College of Medicine, Columbus, Ohio, USA
fTexas Biomedical Research Institute, San Antonio, Texas, USA
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Karen McCoy
eDepartment of Pediatrics, Nationwide Children's Hospital, Columbus, Ohio, USA
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Larry S. Schlesinger
aDepartment of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, Ohio, USA
bThe Ohio State University College of Medicine, Columbus, Ohio, USA
fTexas Biomedical Research Institute, San Antonio, Texas, USA
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Luanne Hall-Stoodley
aDepartment of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, Ohio, USA
bThe Ohio State University College of Medicine, Columbus, Ohio, USA
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DOI: 10.1128/AAC.01782-17
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  • FIG 1
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    FIG 1

    Characterization of M. abscessus smooth (MaSm) and rough (MaRg) variant isolates. (a and d) MaSm and MaRg isolates were distinguishable by colony morphotype on 7H10 agar. (b, c, e, and f) MaRg colonies were also distinguishable by cording at the peripheries of colonies on agar at low magnification (b and e), but not by SEM (c and f). (g) Isolated MaRg and MaSm colonies were grown in 7H9 broth. Growth was similar for the two variants in 7H9 broth with or without Tween 80. (h) MaRg was more aggregative than MaSm. (i) When cultures were removed from shaking after 15 min, MaSm remained suspended, but MaRg rapidly settled out in the absence of Tween. (j) Aggregation was significantly reduced with 0.5% Tween.

  • FIG 2
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    FIG 2

    MaSm and MaRg variants each develop aggregated biofilm structures over time. (a and d) Biomass (expressed as CV absorbance) was greater with MaRg (filled bars) than with MaSm (open bars) (a), and similar results were obtained using the lipophilic probe FM 1-43 to label variants (d). (b and c) Biofilm development did not differ statistically (P > 0.05) between variants when measured by CFU per square centimeter (b) or by mCherry relative fluorescence intensity (RFI) (c). Error bars, standard errors of the means. CFU data represent 3 replicate wells and 3 biological replicates (n = 9); CV and RFI data represent 6 replicate wells and 3 biological replicates (n = 18). (g and j) Pellicle biofilms showed distinct morphologies for MaRg and MaSm variants after 7 days. (e and h) Confocal slices showed levels of mCherry-expressing MaSm (e) and MaRg (h) to be similar. (f and i) The lipophilic probe FM 1-43 showed higher RFI for MaRg (i) than for MaSm (f) (arrows indicate extracellular lipid). (k through n) Finally, orthogonal confocal z-stack images (k and m) and 3-D images pseudocolored to highlight the depth of bacterial biofilms (l and n) showed that complex aggregated biofilm structures were present after 48 h for both variants.

  • FIG 3
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    FIG 3

    Biofilm MaSm or MaRg is more tolerant of hydrogen peroxide than planktonic variants. (a) Planktonic MaSm or MaRg was susceptible to concentrations of H2O2 at or above 1 mM, and MaSm was more susceptible to 10 mM H2O2 than MaRg. (b and c) MaSm or MaRg biofilms were significantly more tolerant of H2O2 at 1 to 10 mM concentrations than planktonic MaSm or MaRg, respectively. (d) MaRg biofilms were more tolerant of H2O2 at concentrations between 5 and 10 mM than MaSm biofilms. Data represent 6 wells per experiment, with 3 biological replicates (n = 18). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

  • FIG 4
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    FIG 4

    Biofilm MaSm or MaRg is more tolerant of low pH than planktonic variants. (a and b) At pH 5.5, the RFIs of planktonic MaSm and MaRg were not significantly different from the RFIs of untreated bacteria. MaSm, but not MaRg, showed a significant difference at pH 4.5. Both showed significant differences at pH 3.5. (c and d) In contrast, MaSm and MaRg showed no statistical difference between untreated biofilms and those treated at pH 4.5 for 2 or 24 h. MaSm and MaRg biofilms treated at pH 3.5 were significantly different from those under all other conditions by two-way ANOVA and were significantly different from each other by a t test (P < 0.001). Data represent 2 experiments with 6 wells per experiment. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

  • FIG 5
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    FIG 5

    Biofilm MaSm or MaRg is more refractory to antibiotic treatment than planktonic variants. (a) Planktonic MaSm showed a significantly lower mCherry RFI than planktonic MaRg in response to amikacin concentrations between 2 and 32 μg/ml. (d) Planktonic MaSm also showed a significantly lower RFI than planktonic MaRg in response to azithromycin concentrations of 4 to 8 μg/ml. (b, c, e, and f) However, biofilms of both M. abscessus variants were significantly more tolerant of antibiotic treatment than planktonic bacteria. (b and c) Concentrations of amikacin that resulted in reduced RFIs for planktonic cells failed to result in significant reductions in the RFIs of biofilms of either variant. (e and f) A similar effect was seen with azithromycin. Data represent 6 wells for each of 2 biological replicates (n = 12). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

  • FIG 6
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    FIG 6

    MaSm or MaRg survives inside THP-1 cells with or without antibiotic treatment. (a and b) Uptake by THP-1 cells infected with opsonized MaSm (open bars) or MaRg (shaded bars) at an MOI of 2.5 for 2 h did not differ significantly between variants as determined by CFU (a) or by microscopy (b). (c to h) Infected-cell monolayers treated with antibiotics had similar intracellular burdens to cells without antibiotic treatment at 48 h. (e and f) Infected-cell monolayers treated with amikacin show that both MaSm and MaRg survive intracellularly in macrophage-like THP-1 cells over 48 h. (g and h) Azithromycin reduced the number of intracellular bacteria; however, MaRg was less susceptible to azithromycin at 48 h. For CFU experiments, data represent 3 biological replicates (5 replicates for no-antibiotic controls) with triplicate wells per experiment. For microscopic analysis, data represent 2 biological replicates (3 for azithromycin) with duplicate plates per experiment. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

  • FIG 7
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    FIG 7

    Biofilm MaSm or MaRg is susceptible to acetic acid. (a and b) Planktonic MaSm or MaRg treated with acetic acid concentrations of 1% or higher was statistically different from untreated MaSm or MaRg by 30 min posttreatment. (c and d) Significantly, this was also observed for MaSm and MaRg biofilms with 2.5% or 5% acetic acid after only 30 min. There was no significant difference between MaSm and MaRg by t test (P > 0.05). Data represent 5 biological replicates with 6 wells per experiment. ***, P < 0.001.

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  • TABLE 1

    CFU reduction

    Antimicrobial agentaLog CFU reductionb under the following growth condition:
    PlanktonicBiofilm
    MaSmMaRgMaSmMaRg
    H2O2 (mM)
        1<1<1<1<1
        5<1<1<1<1
        101.91.2<1<1
        50>7*>7*4.82.3
        100>7*>7*>7*6.2
    HCl (pH)
        5.5NDND<1<1
        4.5NDND<1<1
        3.54.93.42.51.1
    Amikacin (256 μg/ml)NDND<1<1
    Azithromycin (256 μg/ml)NDND00
    Acetic acid (%)
        2-h exposure
            15.341.31.3
            2.5>7*>7*>7*>7*
            5>7*>7*>7*>7*
        30-min exposure
            11<1<1<1
            2.5422.22.9
            5>7*>7*>7*>7*
    • ↵a For all agents or stresses except acetic acid, the exposure time was 24 h.

    • ↵b ND, no data; *, detection limit.

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      Fig. S1

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Mycobacterium abscessus Smooth and Rough Morphotypes Form Antimicrobial-Tolerant Biofilm Phenotypes but Are Killed by Acetic Acid
Gillian Clary, Smitha J. Sasindran, Nathan Nesbitt, Laurel Mason, Sara Cole, Abul Azad, Karen McCoy, Larry S. Schlesinger, Luanne Hall-Stoodley
Antimicrobial Agents and Chemotherapy Feb 2018, 62 (3) e01782-17; DOI: 10.1128/AAC.01782-17

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Mycobacterium abscessus Smooth and Rough Morphotypes Form Antimicrobial-Tolerant Biofilm Phenotypes but Are Killed by Acetic Acid
Gillian Clary, Smitha J. Sasindran, Nathan Nesbitt, Laurel Mason, Sara Cole, Abul Azad, Karen McCoy, Larry S. Schlesinger, Luanne Hall-Stoodley
Antimicrobial Agents and Chemotherapy Feb 2018, 62 (3) e01782-17; DOI: 10.1128/AAC.01782-17
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KEYWORDS

Mycobacterium abscessus
morphotype
biofilm
antibiotic tolerance
acetic acid

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