Swabs were plated onto MacConkey agar (Thermofisher Scientific, Australia) supplemented with 4 µg/ml cefovecin (2013, Zoetis) or ceftiofur (2014–2016) (Sigma-Aldrich, Australia) and incubated overnight at 37 °C. Lactose fermenting colonies were identified, subcultured onto SBA (Thermofisher Scientific, Australia) and incubated at 37 °C overnight. E. coli was then identified from SBA plates by colony morphology, Gram stain and spot indole tests, and identifications were confirmed using MALDI-TOF (Bruker). One representative E. coli colony was subcultured on to SBA and preserved in brain heart infusion broth with 20% glycerol and stored frozen at −80 °C for later analysis. An additional E. coli isolate was selected from the selective agar if two morphologically distinct E. coli colony types were identified on the selective agar plate and on SBA.
Ceftiofur
Manufactured by Merck Group
Sourced in Belgium, Germany, United States
Ceftiofur is an antibiotic product used in the veterinary industry. It is a cephalosporin-class antibiotic that is effective against a variety of bacterial infections in animals.
Automatically generated - may contain errors
Lab products found in correlation
Tetracycline, by Merck Group (5 mentions)
Ammonium formate, by Merck Group (3 mentions)
Florfenicol, by Merck Group (3 mentions)
Amoxicillin, by Merck Group (3 mentions)
Trimethoprim, by Merck Group (3 mentions)
Ampicillin, by Merck Group (3 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Tazobactam, by Merck Group (2 mentions)
Dithioerythritol, by Merck Group (2 mentions)
Cefquinome sodium, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Cefquinome d7, by LGC (2 mentions)
Ceftiofur d3, by LGC (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Potassium hydroxide, by Merck Group (2 mentions)
Sodium tetraborate, by Merck Group (2 mentions)
Neomycin, by Merck Group (2 mentions)
Penicillin g, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
13 protocols using ceftiofur
1
Isolation and Identification of E. coli
2
Preparation of Pasteurized Milk Samples
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Pasteurized whole milk (3.6% fat, 3.3% protein, 5% lactose) was purchased from a local market. To avoid the effect of daily variation of the milk composition, different batches of the product were frozen in 50 mL falcon tubes and stored at −80 °C until further use [21 (link)]. A commercial yogurt starter consisting of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, was purchased from Yogurt Linea (Insao Srl, Milan, Italy). Ceftiofur was purchased from Sigma-Aldrich (Milan, Italy).
3
Analytical Method for Cefquinome and Ceftiofur
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Ceftiofur, cefquinome sodium, dimethylsulfoxide (DMSO) and tazobactam were purchased from Sigma-Aldrich (Overijse, Belgium). Cefquinome-d7, Ceftiofur-d3 and desfuroylCeftiofur were purchased from Toronto Research Chemicals (North York, ON, Canada). Methanol and acetonitrile were of UHPLC grade and were obtained from Fisher Scientific (Erembodegem, Belgium). Water was obtained from a Milli-Q water purification system. Products and solvents used for preparation of buffer solutions and for extraction (acetic acid, formic acid or FA, potassium di-hydrogen phosphate or KH2PO4, potassium hydroxide or KOH, ammonium formate, potassium chloride or KCl, sodium-tetraborate, dithioerythritol or DTE, iodoacetamide) were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (Overijse, Belgium).
4
Quantification of Antibiotics by UHPLC-MS/MS
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Ceftiofur, cefquinome sodium and cefotaxime were purchased from Sigma-Aldrich (Overijse, Belgium). Ceftiofur-d3 and cefquinome-d7, used as isotopically labeled internal standard (IS), were purchased from Toronto Research Chemicals (North York, Canada). Tazobactam was purchased from Sigma-Aldrich (Overijse, Belgium). Methanol and acetonitrile were of UHPLC grade and were obtained from Fisher Scientific (Erembodegem, Belgium). Water was obtained from a Milli-Q water purification system. Products and solvents used for preparation of buffer solutions and for extraction (trichloroacetic acid or TCA, sodium acetate, formic acid or FA, acetic acid, potassium di-hydrogen phosphate or KH2PO4, sodium hydroxide or NaOH, potassium hydroxide or KOH, potassium chloride, dimethylsulfoxide or DMSO, tris(hydroxymethyl)aminomethane or TRIS, ammonium formate, hydrochloric acid or HCl, sodium-tetraborate, dithioerythritol or DTE, iodoacetamide) were HPLC grade and obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (Overijse, Belgium).
5
Antimicrobial Agents Procurement Protocol
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Norfloxacin, ceftiofur (CEFT), florfenicol (FFL), streptomycin sulfate salt (STR), tylosin (TYL), and tetracycline (TCN) were purchased from Sigma-Aldrich (Germany). Antimicrobial Array II kit (EV 3524A) and milk preparation kit (EV 3776) were obtained from Randox Food Diagnostics (UK).
6
Antimicrobial Susceptibility Testing of Dermatophilus congolensis
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Susceptibility testing was performed using the broth microdilution method with Mueller Hinton II broth (REMEL, LEXENA, KS) according to the CLSI guidelines (CLSI VET01 and CLSI VET08) [12 ,13 ]. Broths were incubated at 37 °C and 5% CO2 for 72 h. The control strains were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as well as the reference strain D. congolensis ATCC 14637. All isolates were tested in duplicate with the higher concentration taken as the final in cases when variations occurred. Concentrations tested ranged between 0.125 µg/mL and 64 µg/mL for all antimicrobials. Susceptibility was tested against chlortetracycline, ampicillin, florfenicol, tetracycline, sulfadimethoxine, tylosin, novobiocin, neomycin, amoxicillin, danofloxacin, enrofloxacin, ceftiofur, bacitracin, trimethoprim, penicillin and tulathromycin A (all acquired from SIGMA-ALDRICH, Saint Louis, MO, USA). Differentiation between susceptibility and resistance was performed according to the epidemiological cut off (ECOFF) criteria, or otherwise named the microbiological criterium, using visual estimation (“eye-ball” method) [14 (link)].
7
Staphylococcus Strain Characterization
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Bacterial strains Staphylococcus aureus (ATCC 29213, and two clinical isolates CI 1 and CI 2) and Staphylococcus epidermidis (ATCC 12228, and two clinical isolates CI 1 and CI 2) were procured from Royal University Hospital, Saskatoon, Saskatchewan, Canada. Culture media (Brain-Heart Infusion broth (BHIB) and nutrient broth) and phosphorylcholine were purchased from Thermo Fisher Scientific (Ottawa, ON, Canada). Ceftiofur and all other chemicals used in this study were purchased from Sigma-Aldrich, Canada (Oakville, ON, Canada).
8
Antibiotic Residues Detection in Cow's Milk
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Amoxicillin trihydrate (AMO), ampicillin trihydrate (AMP), oxacillin sodium salt monohydrate (OXA), cloxacillin sodium salt monohydrate (CLO), ceftiofur (CFU), cefalonium hydrate (CFU) and chloramphenicol (CAP) were purchased from Sigma-Aldrich (Steinheim, Germany). Cefazolin CRS (CFZ) and cefapirin sodium CRS (CFP) used were European Pharmacopoeia References. HPLC grade methanol, acetonitrile and water used, whereas acetone and ammonium acetate were of analytical grade and obtained from Sigma-Aldrich (Steinheim, Germany).
Raw cow’s milk was obtained from individual small milk-producing facilities in the regional rural zone of Thessaloniki, Greece. The milk was obtained in the middle of milk production from healthy cows not being treated with antibiotics and was firstly analyzed by the HPLC method so as to be confirmed free of antibiotic residues and sanitizers that could interfere with the kits’ sensitivity. The initial milk sample was prepared in aliquots of 50 mL and was stored at −20 °C until the day of analysis.
Raw cow’s milk was obtained from individual small milk-producing facilities in the regional rural zone of Thessaloniki, Greece. The milk was obtained in the middle of milk production from healthy cows not being treated with antibiotics and was firstly analyzed by the HPLC method so as to be confirmed free of antibiotic residues and sanitizers that could interfere with the kits’ sensitivity. The initial milk sample was prepared in aliquots of 50 mL and was stored at −20 °C until the day of analysis.
9
Antimicrobial Solution Preparation Protocol
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Stock antimicrobial solutions of 1 mg/l (or 1000 units of penicillin G/ml) of penicillin G, tetracycline, doxycycline, ceftiofur and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) were prepared with specific diluents as recommended by Clinical and Laboratory Standards Institute [29 ]. Stock antimicrobial solutions were stored at −20 °C and divided into one-time-use aliquots.
10
Antibiotic Stock Solution Preparation
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The following antibiotics were purchased from Sigma-Aldrich (St. Louis, MO): β-lactams: ampicillin (AMP), penicillin G (PENG), amoxicillin (AMO), cloxacillin (CLO), dicloxacillin (DIC), penicillin V (PENV), oxacillin (OXA), nafcillin (NAP), ceftiofur (CEFT), cefuroxime (CEFM), cefalexin (CEFA), cefoperazone (CEFZ), and cefazolin (CEFL); tetracyclines: doxycycline (DOX), tetracycline (TET), oxytetracycline (OXY), and chlortetracycline (CTE); aminoglycosides: streptomycin (STR), neomycin (NEO), dihydrostreptomycin (DSTR), gentamicin (GEN), kanamycin (KAN), and spectinomycin (SPE); sulfonamides: sulfamonomethoxine (SMM), sulfadimidine (SDM), sulfadiazine (SDZ), sulfamethoxypyridazine (SMP), sulfaquinoxaline (SQX), and sulfamethoxazole (SMX); macrolides: erythromycin (ERY), tylosin (TYL), tilmicosin (TIL), spiramycin (SPI); and lincosamides: lincomycin (LIN), trimethoprim (TMP), and chloramphenicol (CAP). The components used in the preparation of stock solutions and working solutions of antibiotics are shown in Table 1. Stock solutions of antimicrobial agents (1 mg/mL) were prepared in suitable solvents and stored at -20°C in amber glass vials for up to 2 wk. Working standard solutions (1 µg/mL) were prepared by suitable dilutions of the stock solutions until use.
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