Tigecycline

The isolates were initially processed using disk-diffusion antimicrobial susceptibility testing for amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, netilmicin, piperacillin-tazobactam, ticarcillin-clavulanate, and tobramycin with Oxoid (Basingstoke, United Kingdom) disks. Complementarily, an in-house broth microdilution method using cation-adjusted Mueller–Hinton Broth (Sigma-Aldrich, St. Louis, MO, USA) was performed to determine the minimum inhibitory concentration (MIC) for amikacin gentamicin, imipenem, meropenem, colistin, polymyxin B, tigecycline, and ceftazidime-avibactam, and all salts were purchased from Sigma-Aldrich (St. Louis, MO, USA), except for avibactam, which was donated by Pfizer Inc. To complete the antimicrobial susceptibility panel, novel antimicrobials/combinations were evaluated with Liofilchem (Roseto degli Abruzzi, Italy) MIC test strips for ceftolozane–tazobactam, meropenem–vaborbactam, imipenem–relebactam, cefoperazone–sulbactam, cefiderocol, plazomicin, eravacycline, and fosfomycin.

Acinetobacter baumannii 6772166 Muller-Hinton (Oxoid, United States) broth cultures at OD₆₀₀ 0.6 with and without 2.5 μg/ml tigecycline (Sigma-Aldrich, United States) treatment were grown for 30 min at 37°C with shaking. Three biological replicates were prepared. The total RNA of each sample was extracted using the miRNeasy Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions. rRNA was depleted by using the Ribo-Zero Magnetic kit (bacteria) (Illumina, Inc., United States), and converted to cDNA which was sequenced via Illumina HiSeq2500 at Ramaciotti Center for Genomics. Nearly 75 million unique 101 bp reads were obtained from each RNA sample representing more than 310-fold genome coverage. The sequence reads from the 6 RNA-Seq samples (GEO accession no: GSE131451) were aligned against the A. baumannii AB0057 genome and transcriptional coverage for each gene was determined using EDGE-pro (Estimated Degree of Gene Expression in Prokaryotic Genomes) (Magoc et al., 2013 (link)). More than 99% of the RNA-Seq reads of each sample were mapped to AB0057 genome sequence. Genes with significantly different transcription between the control and experimental samples were identified using the DEseq R package (Supplementary Data). Gene differential expression was visualized by metabolic cellular view in Biocyc (Caspi et al., 2016 (link); Karp et al., 2019 (link)).

Drugs were obtained from the following sources: linezolid (MedChemExpress catalog number HY-10394); tedizolid (MedChemExpress catalog number HY-14855); sutezolid (MedChemExpress catalog number HY-10392); sutezolid metabolite SZD-M1 (NIH Reagent Program); delpazolid (MedChemExpress catalog number HY-100180); TBI-223 (MedChemExpress catalog number HY-139398); clarithromycin (Sigma-Aldrich catalog number C9742); azithromycin (MedChemExpress catalog number HY-17506); amikacin (Sigma-Aldrich catalog number PHR1654); tigecycline (Sigma-Aldrich catalog number PZ0021); omadacycline (MedChemExpress catalog number HY-14865); cefoxitin (Sigma-Aldrich catalog number C4786); bedaquiline (MedChemExpress catalog number HY-14881); clofazimine (MedChemExpress catalog number HY-B1046); rifabutin (MedChemExpress catalog number HY-17025), moxifloxacin (Sigma-Aldrich catalog number SML1581); SPR719 (MedChemExpress catalog number HY-12930). All drugs were dissolved in dimethyl sulfoxide at a 10 mM concentration except amikacin, which was dissolved in water.

Antimycin A (#A8674), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, #C2759), chloramphenicol (#C0378-5G), clotrimazol (#C6019), lonidamine (#L4900), paclitaxel (#T7402), potassium cyanide (#60178), rotenone (#45656), sodium azide (NaN_3, #S200), thenoyltrifluoroacetone (TTFA, #88300), tigecycline (#220620097) and vinblastine (VBL, #V1377) were purchased from Sigma (Munich, Germany), cycloheximide (#8682.1) from Roth (Karlsruhe, Germany), N-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone (QVD, #S7311) from Selleckchem (Houston, TX, USA), oligomycin A (#O532970) from Toronto Research Chemicals (Toronto, Canada), etoposide (#1043) from Biovision and staurosporine (STS, #9300) from LC Laboratories (Woburn, MA, USA). All other substances for which a manufacturer is not explicitly specified were obtained from Carl Roth.

AR-12 (OSU-03012) was purchased from MedChemExpress (Monmouth Junction, NJ). Clarithromycin, amikacin, imipenem, cefoxitin, and tigecycline were purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions of AR-12 (128 μg/ml) were dissolved in 32% DMSO (4 μg/ml in 1%). Clarithromycin (2,048 μg/ml) was prepared in 100% DMSO, and other antibiotics at 2,048 μg/ml were prepared in deionized water. All antibiotics were stored as aliquots at –20°C and serially diluted just prior to experimental use.

Tigecycline was purchased from Sigma–Aldrich (St. Louis, MO, USA), while tetracycline was purchased from Carlo Erba (Cornaredo, MI, Italy). Tetracycline was employed as the control to compare the results of Tigecycline effects.

Chloramphenicol (CHL), Erythromycin (ERY), Ciprofloxacin (CIP), Doxycycline (DOX), Rifampicin (RIF), Fusidic acid (FA), Fosfomycin (FOF), Aztreonam (ATM), Clindamycin (CLI), Tigecycline (TGC), Imipenem (IPM), Colistin (CST), Gentamycin (GEN), Piperacillin (PIP), Tazobactam (TZB), Trimethoprim (TMP), Sulfamethoxazole (ST), Daptomycin (DAP), Linezolid (LZD), Oxacillin (OXA), Vancomycin (VAN), and Ceftazidime (CAZ) were purchased from Sigma (Darmstadt, Germany) and Cayman Chemicals (Ann Arbor, MI, USA). All stock solutions of 10 mg/mL were prepared in recommended solvents in non-transparent Eppendorf tubes and kept in −20 °C until use.