The presence of an ESBL and/or AmpC was determined with Cefpodoxime (10μg), Cefotaxime (30μg), Cefepime (30μg) and Ceftazidime (30μg) containing antibiotic discs (Mast Group ltd, UK) by disc diffusion confirmation test. Finally, zones of inhibition were read and recorded on excel sheet and transported to Mast group ESBL/AmpC and CARBA plus calculator spreadsheet and reported as negative or positive for ESBL or/and AmpC.
Antibiotic discs
Manufactured by Mast Group
Sourced in United Kingdom
Antibiotic discs are a type of lab equipment used for antibiotic susceptibility testing. They contain a fixed amount of a specific antibiotic and are placed on a culture medium inoculated with a bacterial sample. The size of the resulting inhibition zone around the disc indicates the susceptibility of the bacteria to the antibiotic.
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8 protocols using antibiotic discs
1
ESBL and AmpC Detection Assay
2
Screening and Confirming ESBL-Producing Isolates
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Presumptive ESBL-producers were screened using cefpodoxime (CPD; 10 μg), cefotaxime (CTX; 30 μg), and ceftazidime (CAZ; 30 μg) antibiotic discs (MAST Group, UK). A 0.5 McFarland standard inoculum of the isolate was spread onto Mueller Hinton Agar (LABM, Lancashire, England) plates. The discs were inserted on the plate, and allowed for incubation at 37 °C for 18–24 h. The breakpoints with zone diameters were evaluated according to the criteria established by.22 The ESBL Set D67C (MAST Group, UK) with a combination of CPD, CTX, and CAZ ± clavulanate (CLA, 10 μg) was used for disc diffusion confirmation test. The disc inserted plates were incubated at 37 °C for 18–24 h. The zone of inhibition was evaluated according to the criteria described by 22 .
3
Antibiotic Susceptibility Testing Protocol
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Antibiotic susceptibility testing of the isolates was carried out by the disc diffusion method, according to the Clinical and Laboratory Standards Institute guidelines (CLSI) (5 (link)). The used antibiotic discs (MAST Group LTD, Merseyside, UK) were: ceftazidime (30 μg), aztreonam (30 μg), carbenicillin (100 μg), piperacillin (100 μg), ticarcillin (75 μg), cotrimoxazole (25 μg), amikacin (30 μg), cefepime (30 μg), ciprofloxacin (5 μg), tobramycin (10 μg), meropenem (10 μg), imipenem (10 μg), cefoxitin (30 μg), and piperacillin/tazobactam (100/10 μg).
4
Antibiotic Resistance Profiling Protocol
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The presence of an ESBL and/or AmpC was determined with Cefpodoxime (10 μg), Cefotaxime (30 μg), Cefepime (30 μg) and Ceftazidime (30 μg) containing antibiotic discs (Mast Group, UK) by disc diffusion confirmation test. After the discs were inserted on inoculated plates, then they were incubated at 35–37 °C for 18–24 h aerobically. Finally, zones of inhibition were read and recorded on excel sheet. The data from the excel sheet was transported to Mast group ESBL/AmpC and CARBA plus calculator spreadsheet (Mast group, UK) and reported as negative or positive for ESBL or/and AmpC and finally the results were recorded.
The results were registered as resistant, intermediate and susceptible; but for the sake of analysis intermediate and resistant isolates were grouped together as resistant. Classification of MDR, XDR and PDR were carried out according to Magiorakos et al, definitions [4 (link)]. All the antibiotic disks were from Oxoid (Oxoid, UK) and Mast discs (Mast group, UK). The inhibition zone diameter was measured using caliper and recorded on excel sheet.
The results were registered as resistant, intermediate and susceptible; but for the sake of analysis intermediate and resistant isolates were grouped together as resistant. Classification of MDR, XDR and PDR were carried out according to Magiorakos et al, definitions [4 (link)]. All the antibiotic disks were from Oxoid (Oxoid, UK) and Mast discs (Mast group, UK). The inhibition zone diameter was measured using caliper and recorded on excel sheet.
5
Antimicrobial Susceptibility Testing of P. multocida
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An antimicrobial susceptibility test was carried Polymerase chain reaction
out by the disk diffusion method in accordance with the guidelines of Clinical and Laboratory Standards Institute (CLSI) [26 ]. In the pipeline for testing, all P. multocida isolates were grown in BHI broth until OD600 reached 0.5 and then were spread over Mueller-Hinton plates (HiMedia). After a few minutes of surface drying, 12 antibiotic discs (Mast group, England) with concentrations as follows: Amikacin (30 μg), amoxicillin (10 μg), ampicillin (10 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamicin (10 μg), kanamycin (30 μg), neomycin (10 μg), ofloxacin (5 μg), enrofloxacin (5 μg), and tetracycline (30 μg) were placed equally-spaced on the surface of the plates and incubated at 37°C for 24 h. The antibiotic susceptibility of isolates was based on the inhibition zone diameter standards provided by the CLSI [26 ]. Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 25923 were used as the quality control strains.
out by the disk diffusion method in accordance with the guidelines of Clinical and Laboratory Standards Institute (CLSI) [26 ]. In the pipeline for testing, all P. multocida isolates were grown in BHI broth until OD600 reached 0.5 and then were spread over Mueller-Hinton plates (HiMedia). After a few minutes of surface drying, 12 antibiotic discs (Mast group, England) with concentrations as follows: Amikacin (30 μg), amoxicillin (10 μg), ampicillin (10 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamicin (10 μg), kanamycin (30 μg), neomycin (10 μg), ofloxacin (5 μg), enrofloxacin (5 μg), and tetracycline (30 μg) were placed equally-spaced on the surface of the plates and incubated at 37°C for 24 h. The antibiotic susceptibility of isolates was based on the inhibition zone diameter standards provided by the CLSI [26 ]. Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 25923 were used as the quality control strains.
6
Disk Diffusion Antibiotic Susceptibility
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Susceptibility testing was performed by disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (23 (link)). Briefly, strains in liquid medium at a 0.5 McFarland standard concentration were grown on Muller–Hinton agar medium and antibiotic discs (Mast Group Ltd., Bootle, UK) was placed on the medium and was incubated for 18–24 h at 37°C and then the zone of inhibition diameter around the disks was measured. Antibiotic tested comprise: [carbapenems: imipenem (IMP: 10 μg), meropenem (MEM: 10 μg)], cefotaxime (CTX: 30 μg), ceftazidime (CAZ: 30 μg), cefepime (CPM: 30 μg), cefoxitin (FOX: 30 μg), ceftriaxone (30 μg), gentamicin (GEN: 10 μg), amikacin (AMK: 30μg), aztreonam (ATM: 30 μg), cephalothin (CET: 30 μg), ciprofloxacin (CIP: 5 μg), augmentin (amoxicillin 20 μg + clavulanic acid 10 μg), trimethoprim/sulfamethoxazole (CMX: 30 μg).
7
Antibiotic Resistance Phenotyping Assay
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The presence of an ESBL and/or AmpC was determined with Cefpodoxime (10 µg), Cefotaxime (30 µg), Cefepime (30 µg) and Ceftazidime (30 µg) containing antibiotic discs (Mast Group ltd, UK) by disc diffusion con rmation test. Finally, zones of inhibition were read and recorded on excel sheet and transported to Mast group ESBL/AmpC and CARBA plus calculator spreadsheet and reported as negative or positive for ESBL or/and AmpC.
8
Kirby-Bauer Antimicrobial Susceptibility Testing
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The Kirby-Bauer method was done to determine antimicrobial susceptibility as recommended by VET04 (CLSI, 2020). For this purpose, a few colonies from 24-hour culture were suspended by a sterile cotton swab into 5 ml saline to prepare 0.5 McFarland concentration (1.5 × 108 CFU/ml). Then, the swab was squeezed in a bottle and cultured on Mueller-Hinton agar (HiMedia, India), and antibiotic discs were placed on it. Ten antibiotic discs (Mast Group, UK), which interested in veterinary were tested compromise ampicillin (10 μg), gentamicin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), enrofloxacin (5μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), lincomycin (2 μg), lincospectin (15.2 μg), oxytetracycline (30 μg) and cephalothin (30 μg). Results were interpreted according to the guidelines of CLSI (2020), and isolates were divided based on their susceptibility profile. The reference strain Escherichia coli ATCC 25922 was used as quality control.
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