The antimicrobial susceptibility testing was carried out for all K. pneumoniae isolates for 12 antibiotics using standard disk diffusion test according to Clinical and Laboratory Standards Institute guidelines (CLSI) (14 ). The antibiotic disks used in the study were imipenem (10 ug), meropenem (10 ug), piperacillin (30 ug), piperacillin-tazobactam (100-10 ug), trimethoprim/sulfamethoxazole (1.25/23.75 ug), ceftazidime (30 ug), cefepime (30 ug), ampicillin-sulbactam (10–10 ug), aztreonam (30 ug), ciprofloxacin (5 ug), gentamicin (10 ug), and tetracycline (30 ug) (MAST Group Ltd, Merseyside, UK). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality controls strains for antimicrobial susceptibility testing.
Aztreonam
Manufactured by Mast Group
Sourced in United Kingdom
Aztreonam is a laboratory product that functions as a synthetic beta-lactam antibiotic. It is used for the detection and identification of specific bacterial strains in a laboratory setting.
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Lab products found in correlation
Ceftazidime, by Mast Group (8 mentions)
Ciprofloxacin, by Mast Group (8 mentions)
Cefepime, by Mast Group (7 mentions)
Imipenem, by Mast Group (7 mentions)
Gentamicin, by Mast Group (6 mentions)
Piperacillin tazobactam, by Mast Group (5 mentions)
Amikacin, by Mast Group (5 mentions)
Cefotaxime, by Mast Group (5 mentions)
Meropenem, by Mast Group (4 mentions)
Piperacillin, by Mast Group (3 mentions)
Ertapenem, by Mast Group (3 mentions)
Imipenem, by Liofilchem (3 mentions)
Ofloxacin, by Mast Group (3 mentions)
Tetracycline, by Mast Group (2 mentions)
E test, by Liofilchem (2 mentions)
Mueller hinton agar (mha), by Merck Group (2 mentions)
Ceftriaxone, by Mast Group (2 mentions)
Tobramycin, by Mast Group (2 mentions)
Levofloxacin, by Mast Group (2 mentions)
Nalidixic acid, by Mast Group (2 mentions)
8 protocols using aztreonam
1
Antimicrobial Susceptibility of K. pneumoniae Isolates
2
Antibiotic Resistance Profiling of NDM-1 Isolates
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Antibiotic susceptibility of the blaNDM-1 positive isolates was determined by the Kirby–Bauer method as recommended by the CLSI. The 11 standard antibiotic disks used include: imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), gentamicin (10 µg), piperacillin/tazobactam (100/10 µg), amikacin (30 µg), ciprofloxacin (5 µg) and aztreonam (30 µg) (Mast Group Ltd, UK). The ESBL phenotype was identified using combined disk method by disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK), applied to all blaNDM−1 positive isolates (15). Moreover, the minimum inhibitory concentrations (MICs) of imipenem (10 µg/ml) [≤ 2 mg/L (susceptible), 4 mg/L (intermediate), and ≥ 8 mg/L (resistant)] (Liofilchem, Roseto degli Abruzzi, Italy) were applied by gradient test strips to blaNDM−1 positive P. aeruginosa isolates [18 ].
3
Antibiotic Susceptibility and ESBL Detection
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The antibiotic susceptibility of all the isolates was tested by employing the Kirby-Bauer’s technique as suggested by the CLSI (10 ). The eleven antibiotic disks used include: imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), cefepime (30 μg), cefotaxime (30 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin/tazobactam (100/10 μg), aztreonam (30 μg) (Mast Group Ltd, UK). Isolates with resistance against a minimum of three groups of antibacterial agents were considered as MDR (11 (link)). To detect ESBL phenotype combined disk method using disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK) was applied to all positively screened isolates by modified hodgE test (MHT) (11 (link)). A growth in the area diameter of ≥5 mm around ceftazidime disc with and without clavulanic acid was expected to be a positive result for ESBL production (12 (link), 13 (link)). The MHT was performed for all isolates as recommended by CLSI (10 ). The E test (imipenem 0.002–32μg/mL) (Liofilchem, Roseto degli Abruzzi, Italy) was applied (according to the manufacturer’s instructions) to all positively screened isolates by PCR test for blaNDM gene, to determine minimum inhibitory concentrations (MICs).
4
Antibiotic Resistance Profiling of Bacterial Isolates
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The resistance pattern of isolates against 15 antibiotics including 5 fluoroquinolones was determined by disc diffusion method on Mueller-Hinton agar (Merck, Germany) as described by the Clinical Laboratory Standards Institute (CLSI 2017) guidelines36 . The antibiotic disks used were as follows: amikacin (30 μg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), gatifloxacin (5 μg), norfloxacin (5 μg), gentamycin (10 μg), imipenem (10 μg), levofloxacin (5 μg), ofloxacin (5 μg), piperacillin (100 μg), piperacillin/tazobactam (100 μg/10 μg), and tobramycin (10 μg) (MAST Co., Berkshire, UK). Drug-resistant patterns were defined as follows: MDR isolates (resistant to at least three antibiotics belonging to different chemical classes), XDR strains (resistant to at least one agent in all but two or fewer antimicrobial groups), and PDR strains (resistant to all antimicrobial classes)37 (link). E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains.
5
Antimicrobial Susceptibility Testing by Disk Diffusion
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The antimicrobial susceptibility test was performed using the disk diffusion method, in Muller-Hinton agar (MHA, Merck, Germany) plate. The antibiotics disc tested based on the minimal growth inhibitory zone diameter were nalidixic acid (NA = 30 μg), ciprofloxacin (Cip = 5 μg), ofloxacin (OF = 5 μg), chloramphenicol (CL = 30 μg), cefotaxime (30 μg), ceftazidime (30 μg) and aztreonam (AZT = 30 μg) (Mast Group, Bootle, UK) [13 (link)]. The results were interpreted as resistant, intermediate or sensitive, in accordance with the guidelines of the CLSI (2015) and the manufacturer protocols [4 (link)]. The strains used for quality control were E. coli ATCC 25922 and Enterococcus fecalis ATCC 29212.
6
Antibiotic Susceptibility Patterns of P. aeruginosa
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At first, the susceptibility pattern of P. aeruginosa isolates to ceftazidime-avibactam (30µg/20µg) and ceftolozane-tazobactam (30µg/10µg) (MAST Co. UK) was assayed using disk diffusion method (Kirby-Bauer). Then, the susceptibility patterns of the ceftazidime-avibactam and ceftolozane-tazobactam resistant isolates to the other antibiotics, including imipenem (10μg), ertapenem (10μg), cefotaxime (30μg), cefoxitin (30µg), ceftazidime (30µg), cefepime (30µg), amikacin (30μg), gentamicin (10μg), ciprofloxacin (5μg), nitrofurantoin (300µg), levofloxacin (10μg), aztreonam (10μg), and fosfomycin (200µg) (MAST Co, UK), were evaluated.10 (link) For performing disk diffusion method, isolates were cultured on Muller-Hinton agar plates (Merck, Germany) and the other procedures were performed according to the CLSI recommendations.11
P. aeruginosa ATCC 27853 was used as a control strain.
After disk diffusion, Minimum Inhibitory Concentration (MIC) of the resistant isolates to both CZA and C/T in disk diffusion was measured. MIC was assessed using the microdilution broth method, according to the recommendations of CLSI.11 Then, MIC of selected antibiotics including imipenem, ceftazidime, amikacin, aztreonam, and ciprofloxacin was evaluated against the resistant isolates to both CZA and C/T. In this assay, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains.
P. aeruginosa ATCC 27853 was used as a control strain.
After disk diffusion, Minimum Inhibitory Concentration (MIC) of the resistant isolates to both CZA and C/T in disk diffusion was measured. MIC was assessed using the microdilution broth method, according to the recommendations of CLSI.11 Then, MIC of selected antibiotics including imipenem, ceftazidime, amikacin, aztreonam, and ciprofloxacin was evaluated against the resistant isolates to both CZA and C/T. In this assay, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains.
7
Antibiotic Susceptibility Profiling of Isolates
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Antibiotic susceptibility of all isolates to ampicillin, amoxicillin-clavulanate, cefoxitin, cefixime, ceftazidime, ceftriaxone, cefotaxime, cefepime, ciprofloxacin, ofloxacin, nalidixic acid, aztreonam, tetracycline, gentamicin, trimethoprim/sulfamethoxazole, nitrofurantoin, and imipenem (Mast Co., UK) was carried out on Muller-Hinton agar (Oxoid Co., UK) using the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI).10
E. coli ATCC 25,922 was used as the quality control strain for antibacterial susceptibility testing. The isolates non-susceptible to ≥1 agent at least three of antibiotic categories were defined as MDR.11 (link)
E. coli ATCC 25,922 was used as the quality control strain for antibacterial susceptibility testing. The isolates non-susceptible to ≥1 agent at least three of antibiotic categories were defined as MDR.11 (link)
8
Antimicrobial Susceptibility Testing for Bacterial Isolates
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Antimicrobial susceptibility testing for the bacterial isolates was carried out using the Kirby-Bauer method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). 15 The following antibiotics were tested: imipenem (10 μg), meropenem (10 μg), doripenem (10 μg), ceftazidime (30 μg), cefepime (30 μg), piperacillin (100 μg), piperacillin/tazobactam (100/10 μg), gentamicin (10 μg), amikacin (30 μg), tobramycin (10 μg), ciprofloxacin (5 μg), aztreonam (30 μg), polymyxin B (300 units), and colistin (10 μg) (Mast Group Ltd, UK). The minimum inhibitory concentrations (MICs) of carbapenems (imipenem [IMI], meropenem [MRP], and doripenem [DOR]) were obtained by an E-test (Liofilchem, Italy), as described in the manufacturer's instructions. Carbapenem resistance was determined based on the MIC breakpoints. When an isolate was resistant to three carbapenems (imipenem, meropenem, and doripenem), that isolate was considered high-level carbapenem resistant. If an isolate was resistant to three or more classes of antimicrobial agents (i.e., penicillins/cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones), that isolate was considered multidrug resistant (MDR). In accordance with the CLSI guidelines, P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as control strains in all susceptibility assays.
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