The minimal inhibitory concentration (MIC) of Enterococcus species to the three fluoroquinolones, including ciprofloxacin, gatifloxacin and levofloxacin (Dalian Meilun Biology Technology Co., Ltd.; Dalian, China) at final concentrations of 0.25–512 mg/L, was determined with the agar dilution method [7 (link)], while E. faecalis ATCC 29212 served as a control isolate. In addition, the MIC of Enterococcus species to ciprofloxacin, gatifloxacin and levofloxacin following treatment with an efflux pump inhibitor reserpine (Dalian Meilun Biology Technology Co., Ltd.; (Dalian, China) at a concentration of 20 mg/L, while the antibiotics-free Mueller-Hinton (M-H) agar (Oxoid, Basingstoke, UK) medium containing 20 mg/L reserpine served as controls.
Ciprofloxacin
Manufactured by Meilun
Sourced in China
Ciprofloxacin is a broad-spectrum antibiotic used in the laboratory setting. It is a fluoroquinolone compound that inhibits bacterial DNA gyrase and topoisomerase IV, essential enzymes for bacterial replication and transcription.
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Lab products found in correlation
Gentamicin, by Meilun (6 mentions)
Levofloxacin, by Meilun (6 mentions)
Meropenem, by Meilun (5 mentions)
Tetracycline, by Meilun (5 mentions)
Imipenem, by Meilun (5 mentions)
Amikacin, by Meilun (3 mentions)
Tigecycline, by Meilun (3 mentions)
Minocycline, by Meilun (3 mentions)
Chloramphenicol, by Meilun (3 mentions)
Ceftazidime, by Meilun (2 mentions)
Polymyxin b, by Meilun (2 mentions)
Ampicillin, by Meilun (2 mentions)
Doxycycline, by Meilun (2 mentions)
Azithromycin, by Meilun (2 mentions)
Rifampicin, by Meilun (2 mentions)
Ofloxacin, by Meilun (2 mentions)
Gatifloxacin, by Meilun (2 mentions)
Erythromycin, by Meilun (2 mentions)
Vancomycin, by Meilun (2 mentions)
Trimethoprim, by Meilun (2 mentions)
12 protocols using ciprofloxacin
1
Fluoroquinolone Susceptibility in Enterococcus
2
Antibiotic Susceptibility Testing of E. coli
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Broth-microdilution and agar-dilution assays were performed to determine the minimum inhibitory concentrations (MICs) of colistin and 10 antibiotics (cefotaxime, ceftiofur, gentamicin, amikacin, ciprofloxacin, meropenem, florfenicol, tetracycline, tigecycline, and piperacillin-tazobactam Meilun Biotechnology Co. Ltd., Dalian) as recommended by the Clinical and Laboratory Standards Institute (CLSI)1 and the European Committee on antimicrobial susceptibility testing (EUCAST).2 Escherichia coli ATCC25922 was used as the quality-control strain. According to a previous report, bacteria resistant to three or more antibiotics were considered to be MDR strains (Zhao et al., 2021 (link)).
3
Antibacterial Agents: Comprehensive Evaluation
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A wide range of clinically-used antibacterial agents including ampicillin, ampicillin-sulbactam (2:1), cefepime, ceftazidime, doxycycline, minocycline, amikacin, gentamicin, ciprofloxacin, levofloxacin, chloramphenicol, imipenem, meropenem, tigecycline, polymyxin B, azithromycin and rifampicin were purchased from Meilun Biological (Dalian, Liaoning, China). Zinc lactate, stannous fluoride and furanone were obtained, respectively, from Yuanye Bio-Technology (Shanghai, China), Macklin Biochemical (Shanghai), and Sigma-Aldrich (St. Louis, Missouri, USA). Bacterial culture media, Muller-Hinton broth (MHB), cation-adjusted MH broth (CAMHB), and tryptic soy broth (TSB) medium were purchased from Haibo Biotechnology (Qingdao, Shandong, China).
4
Reagents and Materials Procurement for Biomolecular Research
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1,4-dioxane, dimethyl sulfoxide (DMSO), dichloromethane (DCM), petroleum ether (PE), ethyl acetate (EA), acetonitrile (MeCN), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium chloride (NaCl), and anhydrous sodium sulfate (Na 2 SO 4 ) were purchased from Tianjin Bohua Chemical Reagent Co. Ltd. (Tianjin, China). Bromelain, tryptone soy agar (TSB), and ciprofloxacin were purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). The PEG 2K -OH, CDCl 3 and DMSO-d 6 were purchased from Beijing Innochem Science & Technology Co, Ltd. (Peking, China). Trifluoroacetic acid (TFA), 4-dimethylaminopyridine (DMAP), (1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HBTU), rhodamine B and glutaric dialdehyde were purchased from Heowns Biochem Technologies LLC. (Tianjin, China). Concanavalin A-tetramethylrhodamine was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China). Gelatin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).
5
Graphene Oxide-based Fluoroquinolone Detection
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Sarafloxacin (SAR), ofloxacin (OFL), gatifloxacin (GAT), enrofloxacin (ENR), ciprofloxacin (CIP), and tetracycline (TC) were purchased from Meilun Biological Technology Corp. (Dalian, China). Dopamine (DA), tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl), and phthalic acid (PA) were obtained from Aladdin (Shanghai, China). Graphene oxide (GO) power was obtained from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). Deionized water (DI) used was prepared by a Milli-Q® ultrapure water system (Bedford, MA USA).
6
Screening Antibiotic Resistance Genes and Potential Inhibitors
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The bacterial strains, plasmids and primers used in this study are listed in Supplementary Table S1, Table S2 and Table S3, respectively. All bacterial strains were grown on Luria-Bertani (LB) plates or in LB broth. All NDM-1-positive strains were originated from our previous studies [17] . E. cloacae 20710 and E. cloacae 20712 were provided by Dr. Yonghong Xiao at Zhejiang University [18] . A. baumannii 21 was provided by Dr. Zhimin Guo at the First Hospital of Jilin University.
The full gene sequences of blaIMP-1, blaVIM-1, blaKPC-2 and blaOXA-48 were synthesized by GeneScript (Nanjing, China) and were inserted into pET28a or pETSUMO for protein expression.
The FDA-approved drug library was purchased from ApexBio Technology (Cat#L1021, Houston, TX, USA). Dexrazoxane, embelin, CAN and NDGA were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Meropenem, amoxicillin, ciprofloxacin, imipenem, erythromycin, chloramphenicol, tetracycline and gentamicin were obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).
The full gene sequences of blaIMP-1, blaVIM-1, blaKPC-2 and blaOXA-48 were synthesized by GeneScript (Nanjing, China) and were inserted into pET28a or pETSUMO for protein expression.
The FDA-approved drug library was purchased from ApexBio Technology (Cat#L1021, Houston, TX, USA). Dexrazoxane, embelin, CAN and NDGA were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Meropenem, amoxicillin, ciprofloxacin, imipenem, erythromycin, chloramphenicol, tetracycline and gentamicin were obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).
7
Antibacterial Agents Procurement and Media Preparation
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Clinically-used antibacterial agents (Magiorakos et al., 2012 (link)), such as piperacillin, ceftazidime, cefotaxime, tetracycline, minocycline, gentamicin, amikacin, ciprofloxacin, levofloxacin, chloramphenicol, imipenem, meropenem, tigecycline, polymyxin B, polymyxin E, vancomycin, bacitracin, and teicoplanin were purchased from Meilun Biological (Dalian, Liaoning, China). In addition, cation-adjusted MH broth (CAMHB), Muller-Hinton broth (MHB) and tryptic soy broth (TSB) medium were obtained from Haibo Biotechnology (Qingdao, Shandong, China).
8
Quantitative Analysis of Antibiotics
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Ciprofloxacin (product number 20130326), sulfamethoxazole (product number 20130406), and trimethoprim (product number 20130609) were purchased from Dalian Meilun Biology Technology Co., Ltd. (Liaoning, China); the internal standard (IS), phenacetin (product number 81105), was obtained from Dr. Ehrenstorfer GmbH (Germany). Ciprofloxacin hydrochloride tablets (250 mg per tablet) were produced by Guangzhou Bai Yun Shan Pharmaceutical General Factory (Guangzhou, China). Compound sulfamethoxazole tablets (sulfamethoxazole 400 mg and trimethoprim 80 mg per tablet) were produced by PKU International Healthcare Group Southwest Pharmaceutical Co., Ltd. (Chongqing, China). Relinqing granules were produced by Guizhou Warmen Pharmaceutical Co., Ltd. (Guizhou, China). High-performance liquid chromatography- (HPLC-) grade acetonitrile and formic acid were supplied by Merck (Darmstadt, Germany). Distilled water was obtained from Watsons Group Co., Ltd. (Hong Kong). All other reagents were of analytical grade and obtained from Kermel Technology Co., Ltd. (Tianjin, China).
9
Antimicrobial Susceptibility Testing of Mycoplasma spp.
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In this study, 11 antimicrobials were used, including: Nemonoxacin (manufactured by Zhejiang Medicine Co., Ltd., Zhejiang, China), macrolides (erythromycin, roxithromycin, azithromycin, and josamycin), tetracyclines (tetracycline, minocycline, and doxycycline), and fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) (Dalian Meilun Biotech Co., China). All agents were obtained in powdered form of known purity and diluted according to their respective manufacturer’s instructions. The broth microdilution method was performed and the minimal inhibitory concentration (MIC) was defined according to Clinical and Laboratory Standards Institutes (Waites et al., 2011 ).
The minimum bactericidal concentrations (MBCs) for nemonoxacin was determined as previously described (Waites et al., 2017a (link)), which included 11 M. pneumoniae isolates, 5 M. hominis isolates, and 5 Ureaplasma spp. isolates. The positive and negative controls consisted of tetracycline (bacteriostatic) and moxifloxacin (bactericidal), respectively. When the MBC was ≥3 dilutions (eightfold) greater than the MIC, the drug was considered bacteriostatic. MBCs that were ≤2 dilutions (fourfold) greater than the MIC were considered bactericidal.
The minimum bactericidal concentrations (MBCs) for nemonoxacin was determined as previously described (Waites et al., 2017a (link)), which included 11 M. pneumoniae isolates, 5 M. hominis isolates, and 5 Ureaplasma spp. isolates. The positive and negative controls consisted of tetracycline (bacteriostatic) and moxifloxacin (bactericidal), respectively. When the MBC was ≥3 dilutions (eightfold) greater than the MIC, the drug was considered bacteriostatic. MBCs that were ≤2 dilutions (fourfold) greater than the MIC were considered bactericidal.
10
Synthesis and Characterization of ZG Compounds and Antibiotic Purchases
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ZG180, (R)- and (S)-ZG197 were synthesized in lab and fully characterized. ONC212 and ADEP 4 were purchased from Topscience and ChemPartner (Shanghai, China), respectively. Antibiotics of vancomycin, spectinomycin, erythromycin, ciprofloxacin, rifampicin, tetracycline, norfloxacin, clindamycin, and gentamicin, were purchased from Dalian Meilun Biotech. Oxacillin was purchased from Sigma-Aldrich. Anhydrous tetracycline (ATC) was purchased from APExBIO.
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