Tetracycline

SMMC-7721 and Bel-7402 cell lines (2 × 10⁷ cells) that constitutively express a tetracycline-controlled transactivator were electroporated with pTRE2hyg–control shRNA plasmid or pTRE2hyg–RIG-I shRNA (RIG-Ish) plasmid using a Gene-Pulser II (Bio-Rad, Hercules, CA, USA) and selected with 0.5 mg/ml of hygromycin B, 0.5 μg/ml of puromycin, and 1 μg/ml of tetracycline (Merck–Calbiochem, La Jolla, CA, USA) for 3–4 weeks to obtain stably transfected cells as described previously [12 (link)]. The core sequences of the two RIG-Ishs, RIG-Ish1 and RIG-Ish2, and control shRNA inserted with flanked miR30a into the pTRE2hyg plasmid vector are shown in Additional file 1: Figure S1a.

The antibiotic susceptibilities of CTX-R E. coli and their transconjugants were determined by disk diffusion method according to CLSI protocols (19 ). The following antibiotic disks were used: ampicillin (10 μg), amoxicillin (20 μg) plus clavulanic acid (10 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), aztreonam (30 μg), imipenem (10 μg), streptomycin (10 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), trimethoprim (5 μg), and sulfonamides (300 μg) (Bio-Rad, Marnes-la-Coquette, France if available or Oxoid, Dardilly, France). The susceptibility breakpoints for all antimicrobials were those recommended by CLSI (20 ).
ESBL production was detected by double-disk synergy test on Mueller-Hinton agar between clavulanic acid and ceftazidime, cefotaxime, or cefepime (20 , 21 (link)). AmpC beta-lactamases detection was based on the inhibitory effect of cloxacillin on AmpC production observed on plates supplemented with 200 mg/L cloxacillin. The control strains used were E. coli ATCC 25922, K. pneumoniae ATCC 700603, and K. pneumoniae CMY-2 from Pr R. Bonnet, France.

Antimicrobial susceptibility testing was performed on all isolates recovered from the two selective media using the disc diffusion method on Mueller–Hinton (MH) agar plates (Neogen, Lansing, Michigan) for ticarcillin (75 μg), amoxicillin/clavulanic acid (20–10 μg), cefotaxime (30 μg), ceftazidime (10 μg), temocillin (30 μg), cefoxitin (30 μg), ertapenem (10 μg), imipenem (10 μg), meropenem (10 μg), ceftazidime/avibactam (10–4 μg), aztreonam (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (SXT) (1.25–23.75 μg), tetracycline (30 μg), amikacin (30 μg), gentamicin (15 μg), and tobramycin (10 μg) (Bio-Rad Laboratories, Algés, Portugal), following EUCAST recommendations and breakpoint tables. Susceptibility to fosfomycin was evaluated by the disk diffusion method (50 μg) on MH agar plates supplemented with 25 μg/mL glucose-6-phosphate, according to EUCAST guidelines [24 ]. Strain E. coli ATCC 25922 was used for quality control. Multidrug resistance was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [25 (link)].

Antibiotic susceptibility testing was performed by the disc diffusion method on Mueller Hinton agar according the standard recommendations of the French Society of Microbiology (CA-SFM, 2010) [8 ]. The following antibiotic discs were tested: ampicillin, amoxicillin+ acid clavulanic, ticarcillin, cefalotin, cefoxitin, cefuroxime, cefotaxim, imipenem, nalidixic acid, aztreonam, norfloxacin, ciprofloxacin, gentamycin, tetracycline, trimethoprim-sulfamethoxazole, streptomycin (Bio-Rad, France). For carbapenems, ertapenem (10 μg/ml) antibiotic discs were used (Bio-Rad, France). The zones of inhibition were measured to assess resistance or susceptibility.

The disk diffusion assay was performed in accordance with the method described by the antibiogram committee of the “French Society of Microbiology” CA-SFM/2014. All isolates were tested for their susceptibility to the following antibiotics: Amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), erythromycin (15 IU), tetracycline (30 IU), gentamicin (10 μg), ciprofloxacin (5 μg), and chloramphenicol (30 μg) (Bio-Rad, France).
From a pure culture of 18-24 h, the bacterial suspension was adjusted to match the 0.5 McFarland turbidity standard. A sterile swab was immersed into the adjusted suspension and then seeded by swabbing onto the entire surface of Mueller-Hinton agar supplemented with 5% defibrinated horse blood and β-NAD (MH-F). After streaking, the inoculum was dried for 5-10 min, and four antimicrobial disks were placed onto the surface of the plate. The plates were incubated at 37°C for 24 h under a microaerophilic atmosphere. Inhibition zones were measured by a caliper, and diameters were interpreted as recommended by the Antibiogram Committee of the French Society of Microbiology [8 ]. C. jejuni ATCC 33560 and C. coli ATCC 33876 were used as control strains.

Disk diffusion method was used to test and confirm the antimicrobial susceptibility of the Enterobacteriaceae isolates using Muller–Hinton agar (MHA, Oxoid, Milan, Italy) and an incubation time of 16–18 h at 37 °C, following the Clinical and Laboratory Standards Institute Guidelines (CLSI) [24 ]. The antimicrobial used were: ciprofloxacin (CIP, 5 µg), nalidixic acid (NA, 30 µg), amoxicillin/clavulanic acid (AMC, 20/10 µg), amoxicillin (AML, 25 µg), levofloxacin (LEV, 5 µg), cefotaxime (CTX, 30 µg), sulphonamides (SSS, 300 µg), tetracycline (TE, 30 µg), trimethoprim/sulphamethoxazole (SXT, 1,25/23,75 µg), trimethoprim (TMP, 5 µg), chloramphenicol (C, 30 µg), and neomycin (N, 30 µg) (Bio-Rad, Marnes la Coquette, France). The results were assessed following the CLSI guidelines [24 ].

The antimicrobial susceptibility was performed by the disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [9 ]. Fifteen antimicrobial agents on Mueller-Hinton agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) were tested: oxacillin, cefoxitin, gentamicin, erythromycin, clindamycin, tetracycline, chloramphenicol, ciprofloxacin, trimethoprim/sulfamethoxazole, fusidic acid, linezolid, rifampicin, tigecycline and vancomycin (Bio-Rad, Marnes la Coquette, France) and penicillin G (Oxoid, Basingstoke, England). The phenotype of resistance to macrolide-lincosamide-streptogramin B was tested and interpreted according to the EUCAST. vancomycin susceptibility was determined with E-test method (bioMerieux, Marcy-l’Etoile, France). Multidrug resistance (MDR) was defined as a resistance to three or more classes of antibiotics.