To test antibiotic resistance in Campylobacter spp., the broth microdilution method was used with 5 % sheep blood. For all other pathogens, antimicrobial susceptibilities were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) Guidelines, 2015 [20 ]. All isolates of Salmonella spp. were tested for their minimum inhibitory concentrations (MICs) of ampicillin, ampicillin-sulbactam, ceftriaxone, cefotaxime, nalidixic acid, ciprofloxacin, levofloxacin, co-trimoxazole, azithromycin, chloramphenicol and tetracycline (Oxoid); DEC were tested for ampicillin, ampicillin-sulbactam, cefotaxime, ciprofloxacin, levofloxacin, chloramphenicol, tetracycline, cefazolin, cefuroxime, imipenem, amikacin and gentamicin (Oxoid); Campylobacter spp. were tested for ciprofloxacin, azithromycin, tetracycline, erythromycin and doxycycline (Oxoid); and Aeromonas spp. were tested for cefotaxime, ciprofloxacin, levofloxacin, co-trimoxazole, chloramphenicol, tetracycline, cefazolin, cefuroxime, imipenem, amikacin and gentamicin (Oxoid). ATCC 25922, 35218, 700603 and 27853 were used as quality control strains. Antibiotic susceptibility was interpreted according to CLSI guidelines, 2015 [20 ].
Levofloxacin
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Germany
Levofloxacin is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a fluoroquinolone antibiotic used in research and scientific applications. The core function of Levofloxacin is to serve as a bactericidal agent, inhibiting the growth and replication of bacterial cells.
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Lab products found in correlation
Ciprofloxacin, by Thermo Fisher Scientific (49 mentions)
Tetracycline, by Thermo Fisher Scientific (36 mentions)
Gentamicin, by Thermo Fisher Scientific (35 mentions)
Amikacin, by Thermo Fisher Scientific (32 mentions)
Imipenem, by Thermo Fisher Scientific (31 mentions)
Chloramphenicol, by Thermo Fisher Scientific (30 mentions)
Erythromycin, by Thermo Fisher Scientific (29 mentions)
Cefepime, by Thermo Fisher Scientific (28 mentions)
Ceftazidime, by Thermo Fisher Scientific (26 mentions)
Meropenem, by Thermo Fisher Scientific (24 mentions)
Cefotaxime, by Thermo Fisher Scientific (21 mentions)
Ampicillin, by Thermo Fisher Scientific (21 mentions)
Ceftriaxone, by Thermo Fisher Scientific (20 mentions)
Vancomycin, by Thermo Fisher Scientific (19 mentions)
Clindamycin, by Thermo Fisher Scientific (18 mentions)
Piperacillin tazobactam, by Thermo Fisher Scientific (18 mentions)
Aztreonam, by Thermo Fisher Scientific (18 mentions)
Trimethoprim sulfamethoxazole, by Thermo Fisher Scientific (18 mentions)
Cefoxitin, by Thermo Fisher Scientific (14 mentions)
Penicillin, by Thermo Fisher Scientific (13 mentions)
88 protocols using levofloxacin
1
Antimicrobial Susceptibility Testing of Pathogens
2
Antibiotics Susceptibility Assay
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A single colony was inoculated in 2 mL LB and incubated with shaking at 250 rpm until OD600nm of 0.5 was reached. Subsequently, a bacterial lawn was spread onto an LB agar plate which contained bovine serum albumin (BSA, Sigma-Aldrich, Burlington, MA, United States) as a control or lactonase (data not reported here). Antibiotic discs (levofloxacin, cefepime, or piperacillin-tazobactam; Thermo Fisher Scientific, Lenexa, KS, United States) were placed in the respective sections of the petri dish and the plates were incubated at 37°C for 48 h. Zone of inhibition (ZOI) was measured to the nearest millimeter (mm) at 24 h (not shown) and 48 h. ZOI standards for the respective antibiotics were evaluated based on Clinical and Laboratory Standards Institute (CLSI).
3
Antibiotic Susceptibility of Group B Streptococcus
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The antimicrobial resistance of GBS was determined through the disk diffusion method (Kirby-Bauer), according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [40 ]. The clinical isolates were tested for susceptibility to seven different antibiotics, including penicillin (10 units), ampicillin (10 μg), cefditoren (5 μg), vancomycin (30 μg), levofloxacin (5 μg), clindamycin (2 μg), and erythromycin (15 μg) (Thermo Fisher Scientific Oxoid Ltd., Basingstoke, UK). Briefly, GBS colonies were suspended in 5 ml of sterile physiological saline, and the turbidity adjusted to a 0.5 McFarland standard, corresponding to a concentration of approximately 1.5 × 108 CFU/ml. A sterile cotton swab was dipped in the bacterial suspension and swabbed over the surface of the Mueller-Hinton agar plates supplemented with 5% defibrinated sheep blood (MHAB) (bioMérieux SA, Marcy-l’Etoile, France). The antibiotic disks were placed on the plates and incubated for 20–24 h at 35 °C in a 5% CO2 atmosphere. After incubation, the zone of inhibition around the disks was measured by a calibrated ruler and interpreted using a standard chart (Table S1 ).
4
Antimicrobial Resistance Profiling of S. pneumoniae
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Antimicrobial resistance testing of the confirmed S. pneumoniae isolates was determined by Etest and Kirby–Bauer disk on Mueller–Hinton agar plates following Clinical and Laboratory Standard Institute guidelines. Antibiotics tested by Etest assay were penicillin and ceftriaxone. Colonies were transferred into test tubes of 5 mL normal sterile saline adjusted to obtain turbidity matching 0.5 McFarland standard. Isolates were inoculated onto Mueller–Hinton agar with sheep-blood plates, and disks impregnated with antimicrobial agents were dispensed on inoculated plates, incubated at 35°C and 5% CO2 for 20–24 hours, and zones of inhibition measured after incubation. For determination of drug resistance, antibiotic-susceptibility breakpoints for S. pneumoniae were according to the 2008 amendments to the Clinical and Laboratory Standard Institute guidelines. Commercial antibiotic disks of amoxicillin (25 µg), cefuroxime (30 µg), cefotaxime (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), levofloxacin (5 µg), linezolid (30 µg), meropenem (10 µg), tetracycline (30 µg), vancomycin (30 µg), and trimethoprim–sulfamethoxazole (25 µg) were purchased from Thermo Fisher Scientific, and penicillin (25 µg) disks were purchased from BioMérieux. The quality-control strain was S. pneumoniae ATCC49619.
5
Antibiotic Susceptibility Testing Protocol
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The antibacterial susceptibility testing was performed usingthe disk diffusion and the VITEK 2 system (bioM’erieux, Marcy l’Etoile, France) and interpreted by the protocol specified in the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2015). The antibacterial agents included cefoxitin, ceftriaxone, cefotaxime, cefepime, ceftazidime, cefazolin, moxifloxacin, ofloxacin, levofloxacin, gentamicin, amikacin, amoxycillin, minocycline, piperacillin, azithromycin, nitrofurantoin, polymyxin B, and meropenem (Thermo Fisher Scientific, USA). MDR isolates were defined as resistant to at least three different classes of antibiotics. Isolates were defined as extensively drug resistant (XDR) if they were not susceptible to at least one agent in all but two or fewer antibacterial categories (ie, bacterial isolates remained susceptible to only one or two antibacterial categories).
6
Antibiotic Susceptibility Testing of Lactobacillus
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Double-layer disc diffusion was used to determine the susceptibility to metronidazole (5μg), clindamycin (2μg), penicillin (2μg) and amoxicillin (10μg; Davies Diagnostics, South Africa), as described previously [58 (link)]. These experiments were performed in duplicate. For rifampicin and rifabutin (Sigma-Aldrich, USA), minimal inhibitory concentrations (MICs) were determined using two-fold serial dilutions according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2019 guidelines, with concentrations ranging from 5–0.00488μg/mL for rifabutin and 25–0.024μg/mL for rifampicin. MICs below the lowest or above the highest concentration tested were assigned a MIC half of the lowest or twice the highest concentration tested, respectively. Experiments were performed in duplicate. For a subset of Lactobacillus strains (n = 20), broader antibiotic susceptibility profiles were determined using Sensititre GPALL1F plates (including ampicillin, cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, daptomycin, erythromycin, gentamicin, levofloxacin, linezolid, moxifloxacin, nitrofurantoin, oxacillin, penicillin, quinupristin/dalfopristin, rifampin, streptomycin, tetracycline, tigecycline, trimethoprim/sulamethoxazole and vancomycin; Thermo Fisher Scientific Inc., USA), according to the manufacturer’s instructions.
7
Antibiotic Susceptibility Testing of Acinetobacter
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Isolates were subjected to Kirby–Bauer disc diffusion sensitivity testing per guidelines of the Clinical and Laboratory Standard Institute (CLSI) with Mueller-Hinton Agar [16 ] and EUCAST [17 ]. The following antibiotics purchased from Thermo Scientific™ Oxoid (United Kingdom) were used: amikacin (30 μg), gentamicin (10 μg), ampicillin (10 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), meropenem (10 μg), amoxicillin/clavulanate (30 μg), cefuroxime (30 μg), ceftazidime (30 μg), cotrimoxazole (25 μg), and nitrofurantoin (300 μg). Acinetobacter isolates with reduced susceptibility to meropenem (10 μg) antibiotic disc (inhibition zone diameter ≤ 14 mm) were selected for carbapenemase and Metallo-beta-lactamase detection (modified Hodge test and combined disc test) as described by Lee et al. [18 (link)] and Yong et al. [19 (link)]. Pseudomonas aeroginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as control strains for quality control. According to the international standard definition for acquired resistance, multidrug-resistant (MDR) phenotype was defined as in vitro nonsusceptibility to at least one agent in three or more categories of antimicrobials [20 (link)].
8
Bacterial Growth Media and Antibiotic Susceptibility Testing
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Bacterial growth media including brain heart infusion agar, Mac Conkey agar, eosin methyl blue agar, sugarsbroth, triple sugar iron agar, urea broth, methyl red media, Voges-Proskauermedia, and Mueller Hinton agar (MHA) were obtained from Oxoid (Hampshire, UK). Paper discs containing standard antibiotics namely ampicillin 10 µg, amoxycillin 25 µg, chloramphenicol 30 µg, cefuroxyme 30 µg, cefotaxime 30 µg, cefoperazone 75 µg, cefepime 30 µg, meropenem 10 µg, amikacin 30 µg, tetracycline 30 µg, ciprofloxacin 5 µg, levofloxacin 5 µg, co-trimoxazole 25 µg, and ceftazidime 30 µg were purchased from Oxoid (Hampshire, UK). Reagents (chrystal violet, 96% ethanol, iodin, safranin O, ammonium oxalate, oksalat, para-dimethylaminobenzaldehyde, butanol, acid chloride, α-naphtol 5%, KOH 40%, and distilled water) were supplied by Microbiology Laboratory, Faculty of Medicine, Universitas Sumatera Utara (Medan, Indonesia).
9
Antibiotic Susceptibility of P. aeruginosa
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Ten antibiotics (purchased from Oxoid Ltd., Basingstoke, UK), namely imipenem (10 µg), meropenem (10 µg), levofloxacin (5 µg), ceftazidime (30 µg), gentamycin (10 µg), amikacin (30 µg), aztreonam (30 µg), piperacillin (100 µg), cefepime (30 µg), and norfloxacin (10 µg), were selected for the antibiotic susceptibility test with P. aeruginosa using the disk diffusion susceptibility method [48 (link)]. Overnight bacterial culture in Tryptic Soy Broth (TSB) (Difco, San Diego, CA, USA) was adjusted to 0.5 McFarland turbidity standards. Subsequently, 0.1 mL of bacterial suspension was spread, using sterile swabs, on Mueller–Hinton agar plates (HI Media Lab. Pvt. Ltd. Ref. M173). Duplicate plates were prepared for each isolate. The antibiotic discs were placed on the agar plates (within 15 min of the inoculation) using sterile forceps, to apply the discs at a distance of 2 cm apart from each other, and incubated for 16–18 h at 37 °C. After incubation, inhibition zones were visible and measured with a ruler, with the measurements recorded in mm [49 (link)].
10
Antibiotic Susceptibility of P. aeruginosa
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The antibiotic profile of fifty P. aeruginosa isolates was investigated using the disc diffusion method [35 (link)]. The tested antibiotics were ciprofloxacin (CIP; 5 μg), gentamicin (CN; 10 μg), imipenem (IPM; 10 μg), cefepime (Feb; 30 μg), levofloxacin (LEV; 5 μg), amikacin (AK;30 μg), piperacillin (PRL; 10 μg), and meropenem (MEL; 5 μg) (Oxoid, UK). The diameter of the clear zone was measured, and the results were interpreted based on the Clinical and Laboratory Standards Institute (CLSI) [43 ].
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