Minimum inhibitory concentration (MIC) values were used to compare relative resistance levels of mutant strains to those of RC85+. Eight β-lactam antibiotics, namely amoxicillin, ampicillin, cefalexin, cefazolin, cefoperazone, cefotaxime, cloxacillin, and methicillin (Sigma-Aldrich) and five other classes of antibiotics, including amikacin, colistin, kanamycin, nalidixic acid, and streptomycin (Sigma-Aldrich) were selected for this. The MIC of each antimicrobial agent was determined using the broth-dilution method in 96-well plates [60 (link)] according to Clinical and Laboratory Standards Institute (CLSI) guidelines, except that cation-adjusted Muller Hinton broth was substituted with LB. The listed MIC values were presented as the mean of three independent experiments.
Cloxacillin
Manufactured by Merck Group
Sourced in United States, Spain, Germany
Cloxacillin is a semi-synthetic penicillin antibiotic used in the treatment of infections caused by penicillin-resistant Staphylococcus aureus. It is a crystalline powder that can be reconstituted for intravenous or intramuscular administration.
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Lab products found in correlation
Ampicillin, by Merck Group (15 mentions)
Amoxicillin, by Merck Group (10 mentions)
Gentamicin, by Merck Group (9 mentions)
Cefazolin, by Merck Group (6 mentions)
Tetracycline, by Merck Group (6 mentions)
Streptomycin, by Merck Group (5 mentions)
Ciprofloxacin, by Merck Group (5 mentions)
Cefotaxime, by Merck Group (4 mentions)
Cefoperazone, by Merck Group (4 mentions)
Cefalexin, by Merck Group (4 mentions)
Kanamycin, by Merck Group (4 mentions)
Penicillin g, by Merck Group (4 mentions)
Vancomycin, by Merck Group (4 mentions)
Oxytetracycline, by Merck Group (4 mentions)
Oxacillin, by Merck Group (4 mentions)
Norfloxacin, by Merck Group (4 mentions)
Methicillin, by Merck Group (3 mentions)
Nalidixic acid, by Merck Group (3 mentions)
Dicloxacillin, by Merck Group (3 mentions)
Nafcillin, by Merck Group (3 mentions)
30 protocols using cloxacillin
1
Antibiotic Resistance Profiling in Mutants
2
Polymeric Drug Delivery System Synthesis
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Acrylamide (AA, 99.5%, CAS: 79-06-1), Methacrylic acid (MAA, 99.5%, CAS: 79-41-4), ethylene glycol dimethacrylate (EGDMA, 99.5%, CAS: 97-90-5), trimethylolpropane trimethacrylate (TRIM, CAS: 3290-92-4), dimethyl sulfoxide (DMSO, reagent grade, CAS: 67-68-5), Malachite green oxylate salt (99.8%, CAS: 2437-29-8), crystal violet (99.8%, CAS: 548-62-9), mordant orange 1 (70%, CAS: 2243-76-7), aluminum oxide (basic, CAS: 1344-28-1), amoxicillin (99.8%, CAS: 26787-78-0), cloxacillin (99.8%, CAS: 61-72-3), and ampicillin sodium salt (99.5%, CAS: 69-52-3) were purchased from Sigma-Merck. Prior to polymerization, all stabilizers were removed from monomers and crosslinkers by passing the compounds over aluminum oxide (basic), thus removing any chemical source that could inhibit the polymerization process. Equipment utilized in the experimentation include a Shimadzu-3600 UV-Spectrophotometer (3.5 mL quartz cuvette, path length of 1 cm), BlueWave 200 UV light source, Leica Microsystem DM300, and a Shimadzu MIRacle10 FT-IR spectrometer equipped with a Zn/Se ATR crystal over an optical range of 4000 to 450 cm−1.
3
MRSA Isolate Characterization and Antibiotic Testing
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The Department of Medical Sciences provided MRSA isolates, including MRSA DMST 20649, 20651, and 20652. In addition, S. aureus ATCC 29213 was acquired from the American Type Culture Collection (ATCC) and used as the quality control strain. Sigma-Aldrich provided all antibiotics employed in this experiment, including cloxacillin (CLX) and nisin (NIS).
4
Synthesis and Characterization of Polymeric Materials
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Ampicillin (AMP), amoxicillin (AMX), oxacillin (OXA), penicillin G (PEN G), penicillin V (PEN V), cloxacillin (CLOX), dicloxacillin (DICLOX), nafcillin (NAFC), and piperacillin (PIPE) were obtained from Sigma-Aldrich (Madrid, Spain). The chemicals used for the polymers synthesis were methacrylic acid (MAA), divinylbenzene 80% (DVB-80), ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM), and the initiator 2,2′-azobis-(2-methylbutyronitrile) (AIMN) from Sigma-Aldrich. MAA, EGDMA, and TRIM were freed from stabilizers by distillation under reduced pressure and AIMN was recrystallized from methanol prior to use. Additionally, DVB was freed from stabilizers by passing through a small column packed with neutral alumina (Aldrich). HPLC grade solvents were supplied by Merck (Madrid, Spain).
5
Antibiotic Susceptibility Testing for ESBL
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The following antibiotic disks from Becton Dickinson were used in the study: aztreonam ATM (30 μg), cefepime FEP (30 μg), ceftazidime CAZ-10 (10 μg) and CAZ-30 (30 μg), imipenem IMP (10 μg), amoxicillin with clavulanate AMC (30/10 μg), ampicillin with sulbactam SAM (10/10 μg), piperacillin with tazobactam TZP (30/6 μg), and ceftazidime with clavulanate CAZ-CL (30/10 μg). The disks containing cefepime with clavulanate FEP-CL (30/10 μg) were purchased from BioRad.
During the determination of ESBL production, two different AmpC inhibitors were used: cloxacillin (Sigma) and boronic acid (purchased as phenylboronic acid from Sigma).
During the determination of ESBL production, two different AmpC inhibitors were used: cloxacillin (Sigma) and boronic acid (purchased as phenylboronic acid from Sigma).
6
Antibiotic Susceptibility Testing Materials
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The antibiotics disks were obtained from MAST (Mast Co., UK). Cloxacillin, cefazolin, clindamycin, and vancomycin powders were obtained from Sigma-Aldrich Company (USA). Mannitol salt agar, Blood agar, Mueller-Hinton agar, DNA agar, Mueller-Hinton broth (MHB), and NaCl were obtained from Merck (Merck Company, USA). Crystal violet, glucose, and agarose were obtained from Sigma-Aldrich Company (USA). U-Bottom 96 well sterile polystyrene microplates were obtained from NEST Co., Ltd, China.
7
Respiratory Syncytial Virus Propagation
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The A549 human epithelial cell line (ATCC CCL-185, Manassas, VA, USA) and HEp-2 human epithelial cell line (ATCC CCL-23, Manassas, VA, USA) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Biochrom AG, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FBS), a cocktail of antibiotics (125 μg/mL ampicillin, 125 μg/mL cloxacillin, and 40 μg/mL gentamicin, Sigma, St Louis, MO, USA), and 1% l -glutamine in 5% CO2 and 37 °C.
This RSV (long Strain, ATCC VR-26) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and provided by Dr. Isidoro Martinez (Centro Nacional de Microbiologia, Instituto de Salud Carlos III). This RSV was propagated in HEp-2 cells as previously described [24 (link),25 (link)] and G2-S16 dendrimer titers were determined by plaque assay in HEp-2 cells.
This RSV (long Strain, ATCC VR-26) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and provided by Dr. Isidoro Martinez (Centro Nacional de Microbiologia, Instituto de Salud Carlos III). This RSV was propagated in HEp-2 cells as previously described [24 (link),25 (link)] and G2-S16 dendrimer titers were determined by plaque assay in HEp-2 cells.
8
Detecting AmpC Overproduction in Isolates
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Isolates were considered as AmpC overproducers when there was at least a twofold dilution difference between each one of the followings: MICs of IMI or MEM, MICs of IMI or MEM plus 250 µg cloxacillin (Sigma-Aldrich, Product Number: 27555) [10 (link)].
9
Comprehensive Analytical Standards Acquisition
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The following solvents, reagents, and antimicrobial agent analytical standards were purchased from Merck (Darmstadt, Germany):
Water was purified using a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany).
The Oasis HLB cartridges (3 mL, 60 mg) were supplied by Waters (Milford, MA, USA).
Methanol and n-hexane (hypergrade for LC-MS LiChrosolv®);
Trichloroacetic acid (TCA) crystals, disodium hydrogen phosphate dihydrate, citric acid monohydrate, and EDTA (for preparing EDTA-McIlvaine buffer solution, pH 4);
Ampicillin, penicillin G, cloxacillin, amoxicillin, penicillin V, oxacillin, dicloxacillin, nafcillin, enrofloxacin, ciprofloxacin, danofloxacin, marbofloxacin, flumequine, tetracycline, 4-epitetracycline, oxytetracycline, 4-epioxytetracycline, chlortetracycline, 4-epichloretracycline, doxycycline, sulfadiazine, sulfathiazole, sulfadimethoxine, sulfadimidine, and enrofloxacin d5 as the internal standards (IS).
Water was purified using a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany).
The Oasis HLB cartridges (3 mL, 60 mg) were supplied by Waters (Milford, MA, USA).
10
Phenotypic Detection of ESBL and AmpC Overproduction
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Extended spectrum β-lactamase (ESBL) production was detected by the double-disc synergy test (DDST) using clavulanic acid-ticarcillin (20/10 mg) and ceftazidime (30 mg) and aztreonam (30 mg) on MH agar as described by Hakemi Vala et al. [10 (link)]. Phenotypic detection of ESBLs can be obscured by the chromosomal AmpC cephalosporinase in P. aeruginosa, hence Cloxacillin-containing DDST was performed. Cloxacillin (250 μg/mL; Sigma, St Louis, MO, USA) was added in MH agar to inhibit cephalosporinase activity [11 (link)].
AmpC overproduction was confirmed according to the method of Rodrίguez-Martίnez et al. [11 (link)]. The isolates were considered as AmpC overproduction when there was at least a twofold dilution difference between the MICs of ceftazidime, imipenem and the MICs of ceftazidime, imipenem plus Cloxacillin [11 (link)].
AmpC overproduction was confirmed according to the method of Rodrίguez-Martίnez et al. [11 (link)]. The isolates were considered as AmpC overproduction when there was at least a twofold dilution difference between the MICs of ceftazidime, imipenem and the MICs of ceftazidime, imipenem plus Cloxacillin [11 (link)].
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