Antimicrobial susceptibility of blaCTX-M harbouring parent strains as well as transformants were determined by Kirby Bauer disc diffusion method and results were interpreted as per CLSI guidelines [17 ]. Following antibiotics were tested: cefotaxime (30μg), cefoxitin (30μg), ceftazidime (30μg), amikacin (30μg), gentamicin (10μg), kanamicin (30μg), ciprofloxacin (5μg), trimithoprim/sulphamethoxazole (1.25/23.75μg), imipenem (10μg), ertapenem (10μg), tigecycline (15μg) and polymyxin B (300 units) (Hi-Media, Mumbai). MIC was also determined for donor strain and transformants against cefotaxime, ceftazidime and ceftriaxone (Hi-Media, Mumbai, India) by agar dilution method.
Ceftazidime
Manufactured by HiMedia
Sourced in India
Ceftazidime is a broad-spectrum cephalosporin antibiotic used in the laboratory setting. It functions as an inhibitor of bacterial cell wall synthesis, thereby demonstrating antimicrobial activity against a range of Gram-negative and some Gram-positive bacteria.
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Lab products found in correlation
Ciprofloxacin, by HiMedia (51 mentions)
Cefotaxime, by HiMedia (49 mentions)
Imipenem, by HiMedia (45 mentions)
Amikacin, by HiMedia (42 mentions)
Gentamicin, by HiMedia (41 mentions)
Ceftriaxone, by HiMedia (33 mentions)
Meropenem, by HiMedia (31 mentions)
Ampicillin, by HiMedia (30 mentions)
Piperacillin tazobactam, by HiMedia (24 mentions)
Cefepime, by HiMedia (23 mentions)
Aztreonam, by HiMedia (20 mentions)
Levofloxacin, by HiMedia (20 mentions)
Cotrimoxazole, by HiMedia (18 mentions)
Cefoxitin, by HiMedia (16 mentions)
Tetracycline, by HiMedia (15 mentions)
Mueller hinton agar (mha), by HiMedia (15 mentions)
Chloramphenicol, by HiMedia (14 mentions)
Nitrofurantoin, by HiMedia (12 mentions)
Nalidixic acid, by HiMedia (12 mentions)
Cefixime, by HiMedia (11 mentions)
79 protocols using ceftazidime
1
Antimicrobial Susceptibility of CTX-M Strains
2
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial sensitivity testing was performed on Mueller-Hinton agar (Hi-Media, Mumbai, India) plates by Kirby Bauer disc diffusion method and interpreted as per CLSI recommendations [26 ]. The antibiotic tested were amikacin (30μg), gentamicin (10μg), netilmicin (30μg), tobramicin (10μg), ceftazidime (30μg), ciprofloxacin (5μg), imipenem (10μg), meropenem (10μg), piperacillin/tazobactum (100/10μg) and polymyxin B (300μg) (Hi-Media, Mumbai, India). MICs of all the isolates were determined by the agar dilution method against cefotaxime, ceftazidime, ceftriaxone (Hi-Media, Mumbai, India), cefepime (Alembic Ltd., Vadodara, India), aztreonam (Aristo Pharmaceuticals Ltd., Mumbai, India), imipenem (United Biotech, Solan, India), meropenem (AstraZeneca Pharmaceuticals Ltd., Bangalore, India), tigecycline (Taj Pharmaceuticals Ltd., Mumbai, India), polymyxin (Celon laboratories Ltd, Andhra Pradesh, India). Escherichia coli ATCC 25922 was used as a control.
3
Isolation and Identification of Antibiotic-Resistant Bacterial Isolates
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The pre-enriched samples were streaked onto MacConkey agar (Sigma-Aldrich) supplemented with cefotaxime and ceftazidime (HiMedia, India) at the concentration of 2 μg/mL each and incubated at 37°C for 18-24 h. One colony from each type was picked from primary growth and subcultured on the MacConkey agar plate supplemented with both cefotaxime and ceftazidime at the final concentration of 1 μg/mL. Purified isolates were identified using the procedures described by Cowan and Steel [26 ]. Biochemically confirmed isolates were stored in trypticase soy broth (TSB; Sigma-Aldrich) containing 30% glycerol at −80°C.
4
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method, adhering to Clinical and Laboratory Standards Institute (CLSI) guidelines [26 ]. The selection of antibacterial disks was based on CLSI 2022 recommendations and availability. The bacterial inoculum, equivalent to 0.5 McFarland standards, was uniformly spread onto Mueller–Hinton agar plates (Himedia India) using a sterile cotton swab applicator. Antibiotic disks for gram-positive bacteria were penicillin G (10 μg), nitrofurantoin (300 μg), ciprofloxacin (5μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), gentamicin (10 μg), cefoxitin (30 μg), and clindamycin (30 μg) (Himedia, India).
The antimicrobial disks for Enterobacteriaceae included piperacillin-tazobactam (100/10 μg), ampicillin (10 μg), nitrofurantoin (300 μg), amoxicillin-clavulanate (20/10 μg), gentamicin (10 μg), cefotaxime (30 μg), ciprofloxacin (30 μg), meropenem (10 μg), piperacillin (100 μg), cefepime (30 μg), amikacin (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), ceftazidime (30 μg), and ceftriaxone (30 μg) (Himedia, India). Antimicrobial disks for Pseudomonas spp and Acinetobacter were gentamicin (10 μg), meropenem (10 μg), ceftazidime (30 μg), piperacillin-tazobactam (100/10 μg), ciprofloxacin (5 μg), and amikacin (30 μg) (Himedia, India).
The antimicrobial disks for Enterobacteriaceae included piperacillin-tazobactam (100/10 μg), ampicillin (10 μg), nitrofurantoin (300 μg), amoxicillin-clavulanate (20/10 μg), gentamicin (10 μg), cefotaxime (30 μg), ciprofloxacin (30 μg), meropenem (10 μg), piperacillin (100 μg), cefepime (30 μg), amikacin (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), ceftazidime (30 μg), and ceftriaxone (30 μg) (Himedia, India). Antimicrobial disks for Pseudomonas spp and Acinetobacter were gentamicin (10 μg), meropenem (10 μg), ceftazidime (30 μg), piperacillin-tazobactam (100/10 μg), ciprofloxacin (5 μg), and amikacin (30 μg) (Himedia, India).
5
Klebsiella pneumoniae Identification and Antibiotic Susceptibility
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The isolates were identi ed by gram stain, standard biochemical methods (urease test, indole test, and carbohydrates fermentation test, motility test, and citrate utilization test) [37] [38], and by K. pneumoniae species-speci c primers (Table 1) targeting the 16S rRNA gene. Antibiotic susceptibility testing was done by the Kirby Bauer disc diffusion method on Mueller Hinton agar using the following antibiotics; cipro oxacin (5mcg), gentamicin (10mcg), ceftazidime (30mcg), imipenem (10mcg), and chloramphenicol (30) (HiMedia Laboratories Pvt. Ltd. Mumbai, India) [39] . E. coli ATCC 25922 and K. pneumonia (ATCC 700603) were used as quality control strains.
Capsule stain was used to detect capsule [40] . String test was used to differentiate between hvKp and cKp strains: if the grown colonies of K. pneumoniae form a string >5 mm in length using a sterile loop, this demonstrates the hypermucoviscosity phenotype [41] .
Capsule stain was used to detect capsule [40] . String test was used to differentiate between hvKp and cKp strains: if the grown colonies of K. pneumoniae form a string >5 mm in length using a sterile loop, this demonstrates the hypermucoviscosity phenotype [41] .
6
Antibiotic Susceptibility Testing of Isolates
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Antibiotic susceptibility tests of all isolates were performed using Kirby Bauer disc diffusion method on Mueller-Hinton Agar with recommended antibiotics by CLSI 2020 guidelines [14 ].The antibiotics used were gentamicin (GEN,30 µg), amikacin (AK, 10 µg), ciprofloxacin (CIP, 5 µg), ceftazidime (CAZ, 30 µg), cefepime (CPM, 30 µg), aztreonam (AT, 30 µg), imipenem (IPM, 10 µg), piperacillin (PI,30 µg), piperacillin-tazobactam (PIT), meropenem (MRP, 10 µg), ofloxacin (OF, 30 µg), Levofloxacin (LEV, 30 µg) and colistin (CL,10 µg) from Hi-Media, Laboratories Pvt. Ltd. India.
Isolates that were non-susceptible to at least one agent in ≥ 3 antimicrobial categories have been categorized under MDR [15 (link)].
Isolates that were non-susceptible to at least one agent in ≥ 3 antimicrobial categories have been categorized under MDR [15 (link)].
7
Antibiotic Susceptibility Testing Protocol
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Antibiotic susceptibility testing was done by Kirby–Bauer disc diffusion method for different classes of antibiotics such as cefotaxime (30 μg), ceftazidime (30 μg), cefoxitin (30 μg), amikacin (30 μg), and ciprofloxacin (5 μg), piperacillin/tazobactam (100 μg/10 μg), and imipenem (10 μg) (Himedia laboratories, Mumbai, Maharashtra, India) as per Clinical and Laboratory Standard Institute guidelines.[11 ] ATCC Escherichia coli 25,922 was used as control strain.
The clinical history was sought for all the K1 and K2 serotypes from the medical records.
The clinical history was sought for all the K1 and K2 serotypes from the medical records.
8
Antibiotic Susceptibility Testing Protocol
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Antibiotic susceptibility testing was performed by modified Kirby Bauer's disc diffusion method on Mueller Hinton Agar medium according to the Clinical Laboratory Standard Institute guidelines (CLSI, 2013) . The antibiotics used were ciprofloxacin ((5 µg), cotrimoxazole (25 µg), cefotaxime (30 µg), ceftriaxone (30 µg), cefixime (10 µg), amikacin (30 µg), gentamycin (10 µg), ceftazidime (30 µg), cefoperazone/sulbactum (75/10 µg), meropenem (10 µg), piperacillin/tazobactam (100/10 µg), chloramphenicol (30 µg) (HiMedia, India). Suspension of bacteria maintained to 0.5 McFarland standards was inoculated on Mueller Hinton Agar (HiMedia, India) plates using sterile swabs, and then antibiotic discs were placed on it. The plates were incubated at 37 °C for 24 hours. The diameter of the zone of inhibition was measured and compared with standard strain. The results were interpreted as sensitive, intermediate, resistant according to CLSI (2013) guidelines. Pseudomonas (ATCC 27853) was used as standard control strains.
9
Antibiotic Susceptibility of E. coli
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All E. coli isolates were tested for antibiotics susceptibility by using the disk diffusion method as reported by Kirby-Bauer [18 (link)]. Briefly, isolates were suspended in sterile 0.85% normal saline and adjusted to 0.5 McFarland standard solution. Then, MHA plates were inoculated, and antibiotic disks were seeded within 15 min after inoculation of MHA plates. MHA plates were incubated aerobically at 37 °C for 16–18 h. The interpretations of zones of inhibitions were performed as recommended by the CLSI 29th Edition guidelines [19 ]. All E. coli that showed intermediate susceptibility to the antibiotics tested were regarded as resistant to such particular antibiotics. Antibiotics tested included ciprofloxacin (CIP 5 μg; HiMedia, Mumbai, India), ampicillin (AMP 10 μg; HiMedia, India), tetracycline (TE 30 μg; HiMedia, India), meropenem (MEM 10 μg; HiMedia, India), ceftazidime (CAZ 30 μg; HiMedia, India), gentamicin (CN 10 μg; HiMedia, India), cefepime (FEP 30 μg; HiMedia, India), and trimethoprim-sulfamethoxazole (SXT 25 μg; HiMedia, India).
10
Antimicrobial Susceptibility Testing
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Amikacin (AMK; 30 μg/disk), ampicillin/sulbactam (SAM, 10/10 µg/disc), cefixime (CFM; 5 µg/disc), cefotaxime (CTX; 30 µg/disc), ceftazidime (CAZ; 30 µg/disc), ceftriaxone (CRO; 30 µg/disc), ciprofloxacin (CIP, 5 µg/disc), gentamicin (GEN, 10 µg/disc), imipenem (IPM, 10 µg/disc), meropenem (MEM; 10 µg/disc), nitrofurantoin (NIT; 300 µg/disc), sulbactam (SUL; 10 µg/disc), trimethoprim/sulfamethoxazole (SXT; 25 µg/ disc) (HiMedia Laboratories, Mumbai,. India), imipenem and sulbactam powder (Sigma-Aldrich Co. St. Louis, MO, USA). The antifungal powders were dissolved in dimethyl sulfoxide (DMSO) and stock solutions diluted based on Clinical and Laboratory Standard Institute (CLSI) guidelines (CLSI M07-A10).
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