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Antibiotics 2020, 9, 98 8 of 12 4.2. Antibiotic Susceptibility Testing Antibiotic susceptibility tests of the strains were carried out by means of the disc diffusion method using Rosco Neo-SensitabsTM tablets commercialized by ROSCO Diagnostica (Taastrup, Denmark). Details of the tested antibiotics and their references are depicted in Table 3. The method was adapted from that described by Kirby-Bauer in 1966 [41]. A few colonies of the strain to be tested were picked from an 18–24 h non-selective agar plate and introduced into a tube containing 4 mL 0.9% NaCl solution obtaining a turbidity equivalent to 0.5 McFarland. One milliliter of this bacterial suspension was then introduced onto a Petri dish containing Mueller-Hinton agar as culture media (Sigma, Saint Louis, United States) and rotated over its entire surface. The surplus suspension was removed using a micropipette. After the inoculum had dried (3–15 min) the antibiotic disks were placed on the agar and the plates were incubated side up in a 35 ◦C incubator. After 16–18 h incubation, the diameters of the zones of complete inhibition were measured to the nearest whole millimeter to classify the strain as susceptible, intermediate or resistant to each specific antibiotic [42]. Classification of the strains as multidrug-resistant was done according to the criteria established by the European Centre for Disease Prevention and Control (ECDC) and the Centre for Disease Control and Prevention (CDC) [28]. Table 3. Antibiotics tested against the E. coli strains. Antibiotic Ampicillin Amoxicillin + Clavulanic acid Cephalothin Cefuroxime Cefotaxime Aztreonam Cefoxitin Ciprofloxacin Nalidixic acid Norfloxacin Azithromycin Tetracycline Gentamycin Fosfomycin Nitrofurantoin Trimethoprim + Sulfamethoxazole 4.3. Photosensitizer and Light Source Dose/μg 10 20 + 10 30 30 30 30 10 5 30 10 15 30 10 200 300 1.25 + 23.7 Reference of the Rosco Neo-SensitabsTM tablets 567NR 60212 567NR 60112 567NR 60612 567NR 60512 567NR 63912 567NR 63612 567NR 62912 567NR 60812 567NR 61412 567NR 76212N 567NR 60312 567NR 62012 567NR 61112 567NR 62312 567NR 62612 567NR 62212 MB was supplied by Panreac (Montcada i Reixac, Spain). It was dissolved in Milli-Q sterile water to give stock solutions with a dye concentration of 78.2 μM. Stock solution was stored in the dark for no more than 1 week and was diluted in PBS immediately before experiments. Red light at 625 ± 25 nm was delivered using an 18 LED light source (Showtec, Par64 Short, Sussex, UK). 4.4. Photodynamic Inactivation of E. coli Growing in Planktonic State: For each strain, three independent tests were carried out as follows: Bacteria were grown aerobically overnight at 37 ◦C in Tryptic Soy Broth (TSB, Panreac 413820.1210, Castellar del Vallès, Spain) in an orbital shaking incubator. A volume of 10 μL of this pre-inoculum was added to 10 mL of sterile TSB and incubated, until the culture reached an optical density of 0.5 at 600 nm (OD600, measured in a Jenway 6305 spectrophotometer), corresponding to ca. 1.5 × 108 CFU/mL. The suspensions were then centrifuged (5 min, 3000 rpm) and the pellet was resuspended in 5 mL of phosphate-buffered saline at pH 4 (PBS, Fisher BP399, New Jersey, USA). Two 0.5 mL aliquots of this bacterial suspension were then diluted with PBS to 1 mL (BS1 and BS2) and two additional 0.5 mL aliquots were diluted with a 62 μM MB solution in PBS also to 1 mL (BS3 and BS4; final MBPDF Image | Effective Photodynamic Inactivation of 26 E coli Strains
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