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

Ma^Sm and Ma^Rg variants each develop aggregated biofilm structures over time. (a and d) Biomass (expressed as CV absorbance) was greater with Ma^Rg (filled bars) than with Ma^Sm (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 Ma^Rg and Ma^Sm variants after 7 days. (e and h) Confocal slices showed levels of mCherry-expressing Ma^Sm (e) and Ma^Rg (h) to be similar. (f and i) The lipophilic probe FM 1-43 showed higher RFI for Ma^Rg (i) than for Ma^Sm (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.

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

Biofilm Ma^Sm or Ma^Rg is more tolerant of low pH than planktonic variants. (a and b) At pH 5.5, the RFIs of planktonic Ma^Sm and Ma^Rg were not significantly different from the RFIs of untreated bacteria. Ma^Sm, but not Ma^Rg, showed a significant difference at pH 4.5. Both showed significant differences at pH 3.5. (c and d) In contrast, Ma^Sm and Ma^Rg showed no statistical difference between untreated biofilms and those treated at pH 4.5 for 2 or 24 h. Ma^Sm and Ma^Rg 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.

Biofilm Ma^Sm or Ma^Rg is more refractory to antibiotic treatment than planktonic variants. (a) Planktonic Ma^Sm showed a significantly lower mCherry RFI than planktonic Ma^Rg in response to amikacin concentrations between 2 and 32 μg/ml. (d) Planktonic Ma^Sm also showed a significantly lower RFI than planktonic Ma^Rg 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.

Ma^Sm or Ma^Rg survives inside THP-1 cells with or without antibiotic treatment. (a and b) Uptake by THP-1 cells infected with opsonized Ma^Sm (open bars) or Ma^Rg (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 Ma^Sm and Ma^Rg survive intracellularly in macrophage-like THP-1 cells over 48 h. (g and h) Azithromycin reduced the number of intracellular bacteria; however, Ma^Rg 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).