Bacterial strains, growth, and reagents.
All mycobacteria strains were grown on Middlebrook 7H9 broth (BD, Franklin Lakes, NJ) supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween 80 (MP Biomedicals, Santa Ana, CA), and 10% ADC enrichment (5% bovine serum albumin [Wako Pure Chemical Industries, Osaka, Japan], 0.81% NaCl, and 2% D-glucose) (7H9-ADC broth) or on Mycobacteria 7H11 agar (BD) supplemented with 0.5% (v/v) glycerol and 10% OADC enrichment (ADC enrichment supplemented with 0.06% [v/v] oleic acid) (7H11-OADC agar).
Hygromycin B (HYG), kanamycin sulfate (KAN), RMP, AMK, and CLA were purchased from Wako Pure Chemical Industries [Osaka, Japan]; INH purchased from SIGMA-ALDRICH [St. Louis, USA]. AMK stock solutions were prepared in water. Stock solutions of all other compounds were prepared in 100% dimethyl sulfoxide (DMSO) and then filter sterilized (pore size 0.45 µm). These components were frozen in aliquots at -20 °C.
CV-AM and SG were purchased from Invitrogen (Life-Technologies Corporation, California, USA). Each aliquot of CV-AM purchased was dissolved in 250 microliters (μl) of 100% dimethyl sulfoxide (DMSO). SG was diluted from the manufacture’s 5 mM stock solution to 50 μM working solution by DMSO. They were frozen at -30 °C until use.
DNaseItreatment and double staining.
Bacterial culture was adjusted to an OD600 of 0.01. Each sample was divided into two tubes of 1 ml each and 1 μl (2 units) DNase I (SIGMA-ALDRICH) added to one of them. Samples were then incubated overnight at 37 °C with rotation 4 rpm. One hundred μl was transferred to a fresh tube. Five μl CV-AM staining solution was added to tubes and incubated for 60 minutes at 37 °C. Then, 1 μl of 0.5 uM SG staining solution was added and incubated for 15 minutes at RT. To remove excess amount of stain the sample was washed with 7H9-ADC once (for Mtb samples fixation was performed using 4% paraformaldehyde/PBS in BSL 3). All samples were diluted in 150 μM NaCl containing 0.05% Tween 80 and analysed by NovoCyte Flow Cytometer [ACEA Biosciences Inc., San Diego, CA, USA] using excitation of 488 (SG) nm and 405 (CV-AM) nm. Control, live, HK, untreated and treated cells by DNase I were also prepared31.
BCG, M. intracellulare, M. avium strains were grown on Sauton media containing KAN and HYG without (-) or with Takara DNase I [Kusatsu, Shiga, Japan (total 50 units)].
All samples were incubated for 5 weeks at 37 °C with weekly addition of DNase I. Then, samples were centrifuged at 2,500 g for 20 minutes to pellet the biomass. The biofilm supernatant was pellet collected and filtered through a 0.45 μm filter and BCG, M. intracellulare and M. avium wet biomass weight.
Bacterial viability after antibiotics treatment.
BCG, M. intracellulare and M. avium were grown until an OD600 of 0.1 in 7H9/ADC broth. The cultures were then diluted in fresh medium to an OD600 of 0.001 with/without DNase I and cultured for a further 72 hours at 37 °C on the rotator. After that bacteria were diluted in fresh medium to an OD600 of 0.001 and treated with INH (0.1 mg/ml), RMP (0.5 mg/ml), AMK (2.5 mg/ml), and CLA (5 mg/ml) for 6 and 24 hours; we also compared control samples without antibiotics. After antibiotics treatment, we prepared ten-fold serial dilutions of each sample from 10−1 to 10−6 and then plated in triplicate on 7H11-OADC agar. The plates were cultured at 37 °C for 3 weeks. The number of colonies were then counted and CFUs (CFU/ml) were calculated.
DNA extraction and electrophoresis.
Bacteria strains were grown to an OD600 of 1.5 in 7H9/ADC broth. Followed by collection of the BCG, M. intracellulare,Mtb, and M. avium pellet by centrifuging the samples at 3500 rpm at room temperature for 20 minutes. Bacterial pellets were suspended in 200 μl of TE lysis buffer [1 M Tris-HCL (pH 8.0) and EDTA (ethylenediaminetetraacetic acid) in Treff tube and frozen at -80 °C. Then, samples were washed with chloroform/methanol, dried, resuspended by 200 ul TE with 20 ul of 1M Tris-HCl pH 9.0 buffers. Next 2.2 ul of 10 mg/mL lysozyme and 5 μl of 10 mg/ml proteinase K (Invitrogen) in the presence of 10% SDS were added. Samples were incubated overnight at 37 °C. All samples were incubated at 50°C for more than 3 hours to lyse the cell wall and membrane. The DNA was then extracted from each sample by addition of an equal volume of 3 M pH 5.2 sodium acetate and phenol: chloroform: isoamylalcohol solution (25:24:1 vol/vol/vol) and mixed gently by inverting the tubes for a few minutes. Next, all the samples were centrifuged for 10 minutes, 15,000 g at RT, and the supernatant containing DNA was transferred to a sterilized Eppendorf tube. Samples were then washed with isopropanol and were centrifuged for 10 minutes above 15,000 g at RT again. After decanting the supernatant, 400 μl of 70% ethanol was added, and the pellet was dissolved. The mixture was centrifuged for 10 minutes at 15,000 g at RT, and the supernatant was decanted gently. After that, RNase A (23 μl of 10 mg/ml; Macherey-Nagel, Germany) was added to DNA samples, and incubated at 37°C for more than 30 minutes. Equal volume of 3 M pH 5.2 Sodium Acetate and phenol: chloroform: isoamylalcohol solution were added and centrifuged, again for 10 minutes with 15,000 g at RT. The supernatant was transferred to a sterilized eppendorf tube, and washed with of chilled isopropanol and 70% alcohol again. The pellet was air-dried and incubated with TE buffer at 50 °C overnight for precipitation and frozen at −20 °C for storage.
The same protocol was used for eDNA extraction, but without the bacterial membrane destruction step. The quality and quantity of the DNA and eDNA samples was assessed using the Agilent 2200 TapeStation [Agilent Technologies, California, USA] was done using gDNA and eDNA36.
Next-generation sequencing of DNA.
eDNA and gDNA samples from bacteria such as BCG, M. intracellulare,Mtb and M. avium were sequenced by MiSeq Illumina sequencer.
Sequencing libraries were prepared using Nextera XT DNA Library Prep Kit (Illumina Inc., San Diego, CA, USA) according to the manufacture’s procedure and purified by AMPure XP beads (Beckman Coulter Inc., Brea, CA, USA). The DNA concentration of each purified library with adapters was measured by QubitTM Fluorometer with Qubit dsDNA HS assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the library size was checked by Agilent 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA). Based on the measured DNA concentration and the size, the molarity of each DNA library was calculated and normalized to 4 nM. Each 4 nM DNA library was pooled and sequenced by the MiSeq system with MiSeq Reagent Kit v3 (Illumina) by following the manufacture’s instructions.
Comparison of gDNA and eDNA.
The genomic sequences and the annotation data of BCG (BCG Tokyo), M. intracellulare (ATCC 13950), Mtb (H37Rv), and M. avium (104) were obtained from the release 40 of Ensemble Bacteria37. The paired-end sequences of gDNA and eDNA samples were filtered using Sam tools39 with maxi parameter of 0.5. The filtered sequences were mapped to each Mycobacterium species genome using BWA38. The number of reads mapped for each gene was calculated using feature counts40. The counts were then normalized by the total read number in a sample, yielding the relative abundance of a gene in a sample. The abundance ratio of eDNA to gDNA was calculated by dividing the relative abundance of eDNA by that of gDNA.
UNIANOVA statistics was performed using IBM SPSS 22.0 software (SPSS, Chicago, IL, USA). Data was analyzed using Games-Howell post-hoc test, Wilcoxon/Kruskal-Wallis non-parametric test, and Student t-test. Differences were considered significant when the P-value was <0.05.