Antimicrobial susceptibility was determined by Kirby–Bauer disc diffusion method13 (link) on MHA plates. The following antibiotics were used: amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (30 μg), trimethoprim/sulphamethoxazole (1.25/23.75 μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg), ceftriaxone (30 μg) and aztreonam (30 μg) (HiMedia). Minimum inhibitory concentrations (MICs) of various antibiotics were determined on MHA plates by agar dilution method according to CLSI guidelines14 using the following antibiotics: cefotaxime, ceftazidime, ceftriaxone, cefepime, imipenem, meropenem, ertapenem and aztreonam (HiMedia).
Imipenem
Manufactured by HiMedia
Sourced in India
Imipenem is a laboratory equipment used for the identification and detection of bacteria. It is a type of carbapenem antibiotic that is commonly used in antimicrobial susceptibility testing. Imipenem is a broad-spectrum antibiotic that is effective against a wide range of gram-positive and gram-negative bacteria.
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Lab products found in correlation
Ciprofloxacin, by HiMedia (53 mentions)
Ceftazidime, by HiMedia (45 mentions)
Gentamicin, by HiMedia (41 mentions)
Amikacin, by HiMedia (41 mentions)
Meropenem, by HiMedia (39 mentions)
Cefotaxime, by HiMedia (34 mentions)
Ceftriaxone, by HiMedia (27 mentions)
Piperacillin tazobactam, by HiMedia (25 mentions)
Ampicillin, by HiMedia (24 mentions)
Cefepime, by HiMedia (21 mentions)
Levofloxacin, by HiMedia (20 mentions)
Aztreonam, by HiMedia (19 mentions)
Cotrimoxazole, by HiMedia (16 mentions)
Cefoxitin, by HiMedia (14 mentions)
Tetracycline, by HiMedia (13 mentions)
Chloramphenicol, by HiMedia (12 mentions)
Ertapenem, by HiMedia (11 mentions)
Mueller hinton agar (mha), by HiMedia (11 mentions)
Ofloxacin, by HiMedia (10 mentions)
Cefixime, by HiMedia (10 mentions)
80 protocols using imipenem
1
Antibiotic Susceptibility Testing Protocol
2
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] .
3
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)].
4
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.
5
Antimicrobial Susceptibility of CTX-M Strains
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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.
6
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).
7
Antimicrobial Susceptibility Testing of Isolates
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Antimicrobial susceptibility of the isolates to the following antibiotics were carried out by the Kirby-Bauer (KB) disk diffusion method according to the Clinical Laboratory Standards Institute (CLSI 2012) guide lines (19 ); tetracycline (TE) [30 μg], amikacin (AN) [30 μg], tigecycline (TIG) [15 μg], colistin (CL) [10 μg], gentamicin (GM) [10 μg], piperacillin (PIP) [100 μg], ciprofloxacin (CIP) [5 μg], ceftazidime (CAZ) [30 μg], cefotaxime (CTX) [30 μg], tobramycin (TOB) [10 μg], amoxicillin + clavulanic acid (AMC) [30 μg], rifampin (Rif) [30 μg], cefixime (CFM) [5 μg], nalidixic acid (NA) [30 μg], imipenem (IMP) [10 μg] and meropenem (MEM) [10 μg] (Hi-media-India). Susceptibility to tigecycline was classified based on EUCAST criteria (20 (link)) (MIC ≤ 0.5 μg/mL; inhibition zone ≤ 17 mm). Minimum inhibitory concentrations (MICs) of the IMP and MEM were determined by E-test (Hi-Media, India) as described by the manufacturer’s instructions and interpreted according to CLSI guidelines (19 ). E. coli ATCC 25922 was used as the quality control strain.
8
MIC Variation Under Gut Conditions
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MIC was determined to check the effect of infection-related in vitro gut conditions, such as bile, osmotic, high and low iron, pH and temperature conditions, on MIC variation in resistant and sensitive isolate by using Ezy MIC™ strips of ampicillin, co-trimoxazole, imipenem, nalidixic acid, ciprofloxacin, tetracycline, nitrofurantoin and chloramphenicol (HiMedia Laboratories Pvt. Ltd., Maharashtra, India). Both the isolates were grown up to mid-exponential phase (MEP) under in vitro gut conditions and swabbed onto Muller Hinton agar (MHA) plates. MIC strips were placed onto plates using an applicator followed by incubation at 37 °C for 16–18 h. The result was read where the ellipse intersects the MIC scale on the strip for the tested antibiotics.
9
Antibiotic Susceptibility of E. coli and Salmonella
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Antibiotic sensitivity testing of isolated E. coli and Salmonella spp. was carried out using the disk diffusion assay as previously described [63 (link)]. Antibiotic classes included fluoroquinolones (levofloxacin, LEV—5 μg; ciprofloxacin, CIP—5 μg), aminoglycosides (gentamicin, GEN—10 μg; streptomycin, S—10 μg), carbapenems (Meropenem, MEM—10 μg; imipenem, IMP—10 μg), amphenicols (chloramphenicol, C—10 μg), macrolides (erythromycin, E—15 μg), and tetracyclines (tetracycline, TE—30 μg) purchased from Hi Media (India). Sensitivity tests were performed on freshly grown isolates having a concentration equivalent to 0.5 McFarland standard using Mueller-Hinton agar media (Hi Media, India). All results were interpreted according to the guidelines provided by Clinical and Laboratory Standards Institute [64 ]. Furthermore, isolates showing resistance against three or more different classes of antibiotics were defined as MDR [65 (link)].
10
Antibiotic Susceptibility Testing for Klebsiella
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Antibacterial susceptibility testing was performed using the disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) documents (13 ). The antibiotics used comprised of piperacillin (100 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefazoline (30 μg), tetracycline (30 μg), kanamycin (30 μg), Imipenem (10 μg), and Meropenem (10 μg) (Himedia, Mombay, India). Minimum inhibitory concentration (MIC) for Imipenem was determined by E-test method (AB Biodisk, Solna, Sweden) for all K. pneumoniae isolates according to CLSI guideline (14 (link)).
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