Fig 1.
Gross morphology and scanning electron microscopy of MAH 104 and A5 7 day-old biofilms.
Log-phase bacteria were resuspended into HBSS and were incubated statically for 7 days at room temperature to allow biofilm formation on an abiotic surface at the surface-liquid interface. (A and B) Gross morphology of a biofilm formed in a 6-well polystyrene plate. (C–F) Biofilms formed on top of coverslips were fixed, labeled with an anti-dsDNA primary antibody, labeled with a secondary antibody conjugated to gold particles, and visualized with scanning electron microscopy. MAH 104 (C) and MAH A5 (D–F) both have biofilm matrix, but the eDNA is only observed in MAH A5 (fibrous structures). Gold particles bound to eDNA, appearing as white spots, were only visualized in MAH A5 biofilms and were localized to the filamentous structures. Asterisks are placed in the micrographs to demonstrate locations of immunogold particles.
Fig 2.
Quantitative assessment of eDNA and CFU in MAH A5 and 104 biofilms.
MAH A5 and 104 static biofilms were formed in 75 cm2 flasks in HBSS at both 22°C and 37°C to represent environmental and host-associated biofilms, respectively. (A and B) After 1 and 7 days of biofilm formation, acellular matrix and supernatant was collected from the biofilms and quantitatively assessed for eDNA using propidium iodide. Bars represent the mean of three biological replicates ± the standard error of the mean (day 7) and the mean of two biological replicates ± the standard error of the mean (day 1). RFU: Relative fluorescence units. PI: Propidium iodide. (C) As biofilms formed over 7 days, wells were mixed thoroughly to remove adhered bacteria and samples were processed for CFU. Day 0 represents the CFU of the inoculum prior to seeding wells for biofilm production. Bars represent the average of three wells of biofilm processed independently ± the standard deviation. The data shown is representative of three biological replicates.
Fig 3.
Survey of eDNA in other MAH strains and nontuberculous mycobacteria.
To assess if eDNA was widespread throughout mycobacteria and not an artifact of MAH A5, biofilms were formed in (A) other M. avium strains and (B) other nontuberculous mycobacteria. Biofilms were formed in 75 cm2 flasks and after 7 days of formation at 30°C, acellular biofilm matrix was extracted and quantified for eDNA. Abbreviations used: M.int = M. intracellulare; M.ab = M. abscessus subsp. abscessus; M.bol = M. abscessus subsp. bolletii; M.ch = M. chelonae; M.mar = M. marinum; M.muco = M. mucogenicum; M.porc = M. porcinum; E = environmental isolate; C = clinical isolate; RFU = relative fluorescence units; PI = propidium iodide.
Fig 4.
Characterization of eDNA in the MAH A5 biofilm.
To compare the similarities of eDNA with genomic DNA (gDNA), side-by-side fingerprinting was done. eDNA extracted from the A5 biofilm matrix was purified and gDNA was extracted from log-phase A5 using a commercial kit. RAPD PCR was conducted from these DNA templates using three sets of random primers that have already been confirmed to work with M. avium.
Table 1.
Sequence results from 7 random pieces of eDNA that were extracted from the MAH A5 biofilm matrix, digested, purified, cloned into a shuttle plasmid, and sequenced.
Fig 5.
Effect of DNase treatment on preventing and removing MAH A5 biofilms.
(A) Biofilms inoculums were made in HBSS supplemented with 1x DNase buffer ± 100 Kunitz units per ml of recombinant DNase I. After 1 day of static biofilm formation at both 22°C and 37°C, supernatants were removed to enumerate the CFU of non-adhered bacteria. (B) For abiotic surface removal, biofilms were formed in 96-well plates in HBSS for 7 days at both 22°C and 37°C. For HEp-2 surface removal, biofilms were formed in RPMI media supplemented with 10% FBS on top of already established HEp-2 monolayers in 96-well plates. At 7 days, supernatants were gently removed and replaced with HBSS supplemented with 1x DNase buffer ± 100 Kunitz units per ml of DNase. Biofilms were incubated for an additional 2 hours and then supernatants removed to enumerate CFU of detached bacteria. For both A and B, bars represent the average of 4 wells of biofilm processed independently ± standard deviation. Data sown are representative of three biological replicates. Statistical comparisons: * P < 0.05; **** P < 0.0001.
Fig 6.
Effect of DNase co-treatment with antimicrobials on the MAH A5 biofilm.
Biofilms were formed in HBSS in 96-well plates and incubated at 22°C and 37°C for 7 days. Supernatants were gently removed and replaced with HBSS containing 16 μg/ml of (A) moxifloxacin or (B) clarithromycin further supplemented 1x DNase buffer ± 100 Kunitz units per ml of recombinant DNase. All biofilms (including 22°C) were incubated for 48 hours at 37°C. Wells were mixed 50 times via pipetting to remove attached bacteria and samples were diluted and plated to obtain total viable bacteria. For both A and B, bars represent the average of 4 wells of biofilm processed independently ± standard deviation. Data shown are representative of three biological replicates. Statistical comparisons: ** P < 0.01; *** P < 0.001.