Strains and plasmid used are listed in Table 2 . Luria-Bertani (LB) medium (Becton Dickinson, Sparks, MD) was used for the growth of all strains. Cultures were incubated at 37 °C and 225 rpm. Kanamycin (kan; 35 μg mL-1; EMD Biosciences, Inc., La Jolla, CA), chloramphenicol (cam; E. coli MIC = 20 μgmL-1: Sigma-Aldrich, St. Louis, MO) and ciprofloxacin (cip; A. baumannii MIC= 0.6 μgmL-1, E. coli MIC= 0.01 μgmL-1; Sigma-Aldrich) and methyl methanesulfonate (MMS; A. baumannii MIC= 25 mM; Sigma-Aldrich) were added to the medium as indicated in the text.
Ciprofloxacin cip
Manufactured by Merck Group
Sourced in United States, Germany
Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antibiotic used in laboratory research and testing. It functions as an inhibitor of bacterial DNA gyrase and topoisomerase IV, which are essential enzymes for bacterial cell division and DNA replication.
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Lab products found in correlation
Vancomycin van, by Merck Group (3 mentions)
Macconkey agar, by BD (2 mentions)
Gentamicin gen, by Merck Group (2 mentions)
Ceftazidime caz, by Merck Group (2 mentions)
Trimethoprim, by Merck Group (2 mentions)
Tetracycline tet, by Merck Group (2 mentions)
Ampicillin amp, by Merck Group (2 mentions)
Luria bertani medium, by BD (1 mentions)
Oxoid, by Thermo Fisher Scientific (1 mentions)
Ceftriaxone cro, by Merck Group (1 mentions)
Tigecycline, by Pfizer (1 mentions)
Colistin cst, by Merck Group (1 mentions)
Anhydrotetracycline atc, by Merck Group (1 mentions)
Levofloxacin, by Merck Group (1 mentions)
Moxifloxacin, by Merck Group (1 mentions)
Sulfaphenazole, by Merck Group (1 mentions)
Ciprofloxacin d8, by Merck Group (1 mentions)
Polymyxin b pmb, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Cyclophosphamide, by Tokyo Chemical Industry (1 mentions)
25 protocols using ciprofloxacin cip
1
Bacterial Growth and Antibiotic Sensitivity
2
Antimicrobial Resistance Profiling of Salmonella Typhi
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All Salmonella Typhi isolates used in this study were collected by the Child Health Research Foundation (CHRF) from the blood of pediatric patients hospitalized at Dhaka Shishu (Children) Hospital (DSH) or pediatric outpatients treated at the Popular Diagnostic Centre (PDC) in Dhaka, Bangladesh. The CHRF team has been preserving Salmonella isolates since 1999 and currently maintains a biobank of over 3500 isolates.
Seventy three S. Typhi isolates were selected from the biobank based on their antimicrobial resistance phenotype (Table2 ). We re-confirmed the identity of all strains using standard biochemical tests and agglutination tests with specific antisera for Salmonella species (Thermo Scientific, Waltham, MA, USA). Antimicrobial susceptibility to ampicillin (amp), cotrimoxazole (sxt) and chloramphenicol (chl) were determined using disk diffusion assays (Oxoid; Thermo Scientific). Broth-microdilution was employed to determine the minimum inhibitory concentrations (MIC) for ciprofloxacin (cip) and ceftriaxone (cro) (Sigma Aldrich, St. Louis, MO, USA). All zone diameters and MIC data were interpreted according to EUCAST v8, 2018 ([67 ], 2018). Metadata for all 73 isolates (sample, organism, year of isolation, setting, patient age [months] and sex) are presented in Additional file 4 : Table S1.
Seventy three S. Typhi isolates were selected from the biobank based on their antimicrobial resistance phenotype (Table
3
Ciprofloxacin Degradation in Water
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Ciprofloxacin (CIP, >99%) was purchased from Sigma-Aldrich, and its physicochemical properties are shown in Table S1.† Pyrite (FeS2) was purchased from Hebei, China. Sodium persulfate, methanol (MeOH), tert-butanol (TBA), ferrous sulfate (FeSO4·7H2O), sulfuric acid, and sodium hydroxide were all analytically pure reagents. All aqueous solutions were prepared using ultrapure water, except for river water (Uma River in China) and tap water (laboratory) (Table S2† showed physicochemical properties of river water and tap water).
4
Antibiotic Susceptibility Testing of C. difficile
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Minimum inhibitory concentrations (MIC) for MTZ (Sigma), VAN (Sigma), ciprofloxacin (CIP) (Sigma), piperacillin/tazobactam (TZP) (Sandoz), and tigecycline (TIGE) (Pfizer) were determined by the agar dilution method and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [23 ]. Briefly, TY agar plates were used for susceptibility testing in order to mimic the conditions of the sporulation assays. A 10-μl sample from a log-phase culture of C. difficile (optical density at 600 nm = 0.5) was streaked over TY agar plates containing doubling dilutions of each antibiotic. Plates were incubated under anaerobic conditions for 48 h and MIC values were determined as the antibiotic concentration where colonies did not grow.
5
Antimicrobial Susceptibility Profiles of APEC
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Initially, antimicrobial susceptibility profile was established for all the tested APEC serotypes using cation-adjusted Muller-Hinton broth (CAMHB) micro-dilution method according to clinical and laboratory standards institute (CLSI) guidelines20 . E. coli ATCC 25922 was used as quality control strain. Four antimicrobials; ampicillin (AMP, #A9518 Sigma-Aldrich), ciprofloxacin (CIP, #17850 Sigma-Aldrich), colistin (CST, C4461 Sigma-Aldrich), and tetracycline (TET, T7660 Sigma-Aldrich) that are currently used in poultry industry and belonging to different classes of antimicrobials; penicillins, quinolones, polymixins, and tetracyclines, respectively were evaluated for susceptibility according to their MIC breakpoints for resistance (AMP ≥ 16 µg/mL, CIP ≥ 4 µg/mL, CST ≥ 4 µg/mL, and TET ≥ 16 µg/mL)21 (link). To determine the effect against antimicrobial resistant APECs, the MIC and MBC of cidal SMs were compared between the antimicrobial susceptible and resistant APEC serotypes.
6
Ciprofloxacin Antibiotic Preparation
7
Synergistic Lethality of Resistance Mechanisms
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To evaluate the synergistic lethality of a murA knockdown combined with OXA-23 overexpression, ATCC 17978 Tn7::dCas9 pJE53::murA-sgRNA was electroporated with pQF1266-hyg vector and pQF1266-hyg::blaOXA-23 and selected for on LB plus Kan plus Hyg. Well-isolated transformants were picked and resuspended in 50 μl LB. Resuspensions were seriallydiluted and spotted onto LB plus KAN plus HYG agar with increasing concentrations of anhydrotetracycline (ATc; Sigma-Aldrich) and incubated overnight at 37°C.
To evaluate the synergistic lethality of peptidoglycan synthesis chemical inhibition combined with OXA-23 overexpression, ATCC 17978 Tn7::dCas9 was electroporated with pQF1266-hyg vector and pQF1266-hyg::blaOXA-23 and selected for on LB plus Hyg. Well-isolated transformants were picked and resuspended in 50 μl LB. Resuspensions were serially diluted and spotted onto LB plus HYG agar with increasing concentrations of fosfomycin (FOF; Sigma-Aldrich),d -cycloserine (Dc; Sigma-Aldrich), or ciprofloxacin (CIP; Sigma-Aldrich) and incubated overnight at 37°C.
To evaluate the synergistic lethality of peptidoglycan synthesis chemical inhibition combined with OXA-23 overexpression, ATCC 17978 Tn7::dCas9 was electroporated with pQF1266-hyg vector and pQF1266-hyg::blaOXA-23 and selected for on LB plus Hyg. Well-isolated transformants were picked and resuspended in 50 μl LB. Resuspensions were serially diluted and spotted onto LB plus HYG agar with increasing concentrations of fosfomycin (FOF; Sigma-Aldrich),
8
Quinolone Antimicrobial Evaluation
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The quinolones used in this study were: ozenoxacin (OZN, Ferrer Internacional S.A. Barcelona, Spain), moxifloxacin (MOX), levofloxacin (LVX) and ciprofloxacin (CIP) obtained from Sigma-Aldrich (St. Louis, MO, USA).
9
Detecting APEC Virulence Genes in Ciprofloxacin-Resistant E. coli
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The enriched modified E. coli was streaked onto MacConkey agar (BD Biosciences, Sparks, MD) containing 4 μg/mL of ciprofloxacin (CIP ; Sigma-Aldrich, St. Louis, MO). Subsequently, suspected E. coli colonies were identified by PCR as previously described (Candrian et al., 1991 (link)). Confirmed E. coli were also analyzed for 5 genes (iroN, ompT, hlyF, iss, and iutA) as the minimal predictors of APEC virulence described by Johnson et al. (2008) (link). If isolates from the same farm showed the same antimicrobial susceptibility patterns, one isolate was randomly selected. A total of 33 FQ-resistant APEC isolates were included in this study.
10
Synthesis and Characterization of 2Abz23S29
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The test peptide 2Abz23S29 with sequences of (N-RYGTC(Acm)IYQ2AbzRLWAFS-C) was synthesized by biomatik (Canada) in trifluoroacetate (TFA) salt, and high-performance chromatography (HPLC) purified to > 96% with water and acetonitrile. 2Abz23S29 was dissolved in distilled water and was stored in dark at −80°C until use. Ciprofloxacin (cip) was also used in this study (Sigma-Aldrich). Stock solution from dry powder was prepared and stored in dark at −20°C until use.
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