Nocardia spp. isolates and ATCC strain
Nocardia spp. isolates from the Collection of Cultures of the Laboratory of Medical Research in Mycology (LIM53) were included in this study (n = 15; Table 1). Eight isolates were identified in a previous study [4] and the other seven Nocardia beijingensis(N. beijingensis)-1180 (MW348983), N. beijingensis-1181 (MW348984), Nocardia brasiliensis(N. brasiliensis)-95 (MW348931), N. brasiliensis-129 (MW348933), N. brasiliensis-519 (MW348935), Nocardia farcinica(N. farcinica)-788 (MW348936), and N. farcinica-2901 (MW348955) were identified by Gram and Ziehl Neelsen staining methods, morphological characteristics and molecular sequencing. A 606 bp fragment of the 16S rRNA gene was amplified with the primers Noc1 (5′-GCTTAACACATGCAAGTCG-3′) and Noc2 (5′-GAATTCCAGTCTCCCCTG-3′), and sequenced as previously described [18]. The species was confirmed by comparing them against type strain sequences with the BLAST algorithm v.2.2.10 (http://www.ncbi.nlm.nih.gov/BLAST). Similarity values of ≥98.0% for 16S rRNA were deemed to indicate the same species.
CLSI-recommended quality control ranges for Amikacin (AMK), Ciprofloxacin (CIP), Minocycline (MIN) and Trimethoprim-Sulfamethoxazole (TMP-SMX) were performed by using a strain of Staphylococcus aureus (S. aureus ATCC 29213) [7]. Nocardia spp. isolates were cultured on Tryptone Soy Agar (TSA; Kasvi, Italy) at 35 °C for 7 days and the ATCC strain was also cultured on TSA at 35 °C for 24 h.
Resazurin solution
The 0.01% Resazurin solution was prepared by dissolving the powder dye (Sigma-Aldrich, Munich, Germany) in 1× PBS, pH 7.2 (Life Technologies, Carlsbad, CA, USA). After complete dissolution, the solution was sterilized by a 0.22 μm membrane filtration, stored at 4 °C, protected from light, and used within 3 days.
Antimicrobial solutions
The antimicrobial solutions were prepared as recommended by the CLSI document M100-S24 [19]. Briefly, each antimicrobial agent (all from Sigma-Aldrich, Munich, Germany) was dissolved and diluted separately: AMK and MIN were dissolved and diluted in water; CIP was dissolved in Hydrochloric Acid 0.1 N and diluted in water; TMP was dissolved in Lactic Acid 0.05 M and diluted in warm water; and SMX was dissolved in Sodium Hydroxide 2.5 M and diluted in water. The drugs were two-fold serially diluted in CAMHB in 96-well microplates (100 μL/well; flat-bottomed cell culture microplates from Corning, Durham, NC, USA) at the following concentration ranges: 0.5–256.0 mg/L for AMK, 0.12–64.0 mg/L for CIP, 0.06–32.0 mg/L for MIN, and 0.12–64.0/2.37–1216.0 mg/L for TMP-SMX. The microplates were frozen at − 80 °C until use.
Antimicrobial susceptibility test
The MICs were determined by the BMD method, according to CLSI document M24 for Nocardia [7]. Briefly, inoculum suspensions for each Nocardia isolate were prepared by dissolving the bacterial content with saline and glass microbeads (Sigma Aldrich, Munich, Germany), vortexing vigorously and then allowing it to precipitate for 10 to 15 min. The concentration was adjusted by dilution on Cation-Adjusted Mueller-Hinton Broth (CAMHB; Becton Dickinson and Company, Sparks, MD, USA).
Medium control wells had 200 μL/well of drug-free CAMHB, and inoculum control wells had 100 μL/well of drug-free CAMHB and 100 μL/well of bacterial inoculum. All the other wells were inoculated with 100 μL/well of bacterial inoculum on microplates with antimicrobial solutions, previously prepared. Microplates were incubated in a moist chamber at 35 °C.
Following incubation of 24 h, 20 μL/well of 0.01% sterile resazurin solution were added to all wells and microplates were re-incubated for 48 h for colour development. A change in colour from blue to pink indicated bacterial growth.
The ATCC strain inoculum suspension was prepared as described for Nocardia isolates, except for the incubation period. After incubation for 17 h, 20 μL/well of 0.01% sterile resazurin solution were added to all wells and the microplates were re-incubated for more 3 h for colour development.
Following the stablished incubation periods for the ATCC strain (17 and 20 h) and for Nocardia isolates (48 and 72 h), the microplates were evaluated by the Gold Standard visual reading without any dye, the visual reading with Resazurin, or by OD readings without any dye, at 560 nm in the Versa Max Microplate Reader spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
Quality control
Growth in medium control wells indicated contamination or absence of growth in inoculum control wells indicated unviability of the isolate, and the corresponding assay was discarded. At least two wells were matched among the triplicates for a valid result.
Disk diffusion test
The Disk Diffusion test was performed with the isolates suspensions, prepared as described on M24 CLSI document for Nocardia [7], and plated on 90-mm-diameter Mueller-Hinton agar plates (DME, Araçatuba, SP, Brazil). Antimicrobial disks of TMP-SMX (25.0 μg; Sensidisc, DME, Araçatuba, SP, Brazil) were then placed on the agar, and the plates were incubated at 35 °C for 3 to 5 days. Interpretive categories of Disk Diffusion results for Nocardia spp. isolates were based on the M24 CLSI document: a zone of ≥35 mm indicated susceptibility, and a zone of ≤15 mm indicated resistance.
Analysis
For the visual interpretation of the results, the MIC well was determined by its comparison with medium and inoculum drug-free control wells. The MIC well was the one with the lowest drug concentration that prevented the bacterial growth and the colour change within the incubation period, being similar to the medium control well (no visible growth and blue with Resazurin). The inoculum with drug and the inoculum control drug-free wells should show visible growth and should be pink with resorufin. For the spectrophotometric readings, the percentages of bacterial growth were calculated with the following eq. [20]: bacterial growth percentage (%) = (OD from the inoculum with drug well - the OD from the medium control well) / (OD from the inoculum control drug-free well - OD the OD from the medium control well) × 100. The MIC well was the one with the lowest drug concentration with ≤10% of bacterial growth [14].
The Pearson Correlation Coefficients (Pearson’s r) and respective 95% Confidence Intervals (95% CI) were determined by the Prism 5.01 statistical software (GraphPad, San Diego, CA, USA).