Spiramycin

A summary of the chemical structures of the compounds used in this study is available in Fig. 2. Azithromycin 1, erythromycin 9, erythromycin oxime 11 and dirithromycin 13 were obtained commercially from Ak-Scientific (Union City, CA, USA). Roxithromycin 12, clindamycin 16 and spiramycin 15 were from Sigma Aldrich (St Louis, MO, USA). 6-O-Megosaminyl erythromycin A 14 was a gift from C Goodman, E Rodriguez and H Gramajo [41 (link)]. Compounds 4–8 were provided by GlaxoSmithKline (Tres Cantos, Spain). Synthesis of these compounds are as previously described: 4 (12e) and 5 (1j) [44 (link)]; 6 (11c) [42 (link)]; and 7 (7/47) and 8 (7/25) [43 (link)]. AZR-desclad 2 was prepared according to Istuk et al. [65 (link)]. ERY-desclad 10 was prepared following methods of LeMahieu et al. [66 (link)]. AZR-desglycan 3 was prepared following similar procedures previously outlined in the literature [67 , 68 ]. In general, compounds were solubilized with ethanol as vehicle, with the exception of the initial characterization of azithromycin (1), where both ethanol and DMSO were compared, and compounds 4–8 (DMSO), owing to the limited amount of material available.

The antibiotics used in this study are: Ampicillin (A1593, Sigma-Aldrich), Carbenicillin (C3416, Sigma-Aldrich), Cefotaxime (454950050, Acros Organics), Cefoxitin (C4786, Sigma-Aldrich), Chloramphenicol (C0378, Sigma-Aldrich), Ciprofloxacin (F17850, Sigma), Clindamycin (21462-39-5, RPI), Doxycycline (D9891, Sigma-Aldrich), Erythromycin (E5389, Sigma-Aldrich), Fusidic acid (F0881, Sigma-Aldrich), Gentamycin (PRX1002-Premier Pro RX), Kanamycin (60616, Sigma-Aldrich), Levofloxacin (28266, Sigma-Aldrich), Lomefloxacin (L2906, Sigma), Meropenem (NDC6332350720, Fresenius Kabi LLC), Nitrofurantoin (N7878, Sigma-Aldrich), Oxacillin (NDC25021-162-24, Sagent Pharmaceuticals), Penicillin (P8396 Sigma-Aldrich), Piperacillin/tazobactam (NDC60505-0688-4, Apotex Corp.), Rifampicin (R3501, Sigma-Aldrich), Spectinomycin (85555, Sigma-Aldrich), Spiramycin (S9132, Sigma-Aldrich), Sulfamonomethoxine (32091, FLUKA), Tetracycline (87128, Sigma-Aldrich), Tobramycin (T4014, Sigma-Aldrich), Trimethoprim (T7883, Sigma-Aldrich), Vancomycin (NDC67457-340-00, Mylan). All antibiotic solutions were prepared by following manufacturers’ instructions.

Twenty investigated antibiotics—enrofloxacin (ENR), erythromycin (ERY), levofloxacin (LVX), norfloxacin (NFX), oxytetracycline (OTC), sulfamethazine (SMZ), sulfamethoxazole (SMX), sulfaquinoxaline (SQX), trimethoprim (TMP), ciprofloxacin (CPR), metronidazole (MTZ), roxithromycin (RXM), clarithromycin (CLR), flumequine (FMQ), enoxacin (ENX), tylosin tartrate (T-T), midecamycin (MED), spiramycin (SPR), josamycin (JOS), azithromycin (AZM) (Supplementary Table 1) were purchased from Sigma Aldrich (Buchs, Switzerland). LC/MS-grade methanol, acetonitrile and formic acid were purchased from Carlo Erba Reagents (Val-de-Reuil, France), ultrapure water (>18.2 MΩ cm^–1) was prepared with the Milli-Q IQ 7000 system (Millipore SAS, Molsheim, France). Individual raw solutions (200 mg/L) were prepared for each antibiotic by dissolving the standard powder in methanol and stored in the dark at −20°C. The standard solution mixtures were prepared at three concentrations (10, 5, 1 mg/L) by diluting the raw solutions with water and then stored in the dark at 4°C. Antibiotics were measured in all the collected biofilms by the procedure described in Aubertheau et al. (2017) (link).

β–lactam: penicillin G (PEN), cefquinome (CEF); aminoglycosides: neomycin (NEO), streptomycin (STR); tetracyclines: doxycycline (DOX), tetracycline (TET); macrolides: erythromycin (ERY), spiramycin (SPI); sulfonamides: sulfadiazine (SDZ), sulfadimidine (SDM); lincosamides: lincomycin (LIN); quinolones: danofloxain (DAN), enrofloxacin (ENR); trimethoprim (TMP); and chloramphenicol (CAP) were all purchased from Sigma-Aldrich (St. Louis, MO, United States). Drugs for the preparation of antimicrobial solutions were stored and handled according to the manufacturers’ instructions before use. In addition, the methods for the preparation of stock solutions and working standard solutions of antibiotics were shown in Table 1.

Spiramycin was purchased from Sigma-Aldrich, Germany. Preparation, dose, and methods of administration were as previously described [38 ].

Spiramycin, LPS from Escherichia coli, MTT, dimethyl sulfoxide (DMSO), Griess reagent, sodium nitrite, protease inhibitor cocktail, and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cytoplasmic extraction reagents (NCER), Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin/streptomycin solution), and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Enzyme-linked immunosorbent assay kits for prostaglandin E₂ (PGE₂), IL-1β, interleukin-6, and TNF-α were purchased from R&D Systems Inc. (Minneapolis, MN, USA). Antibodies against β-actin, anti-iNOS, anti-COX-2, p65 (Ser536), p-p65 (Ser32), and anti-NF-κB (IκBα) were purchased from Calbiochem (San Diego, CA, USA). T-ERK, P-ERK, T-JNK, P-JNK, T-P38, and P-P38 were obtained from Cell Signaling Technology (Beverly, MA, USA). All other reagents were of analytical grade.