For initial screening, antimicrobial susceptibility testing was performed with 16 antimicrobial agents (HiMedia Laboratories Mumbai, India) namely; norfloxacin (10 μg), cefotaxime (30 μg), imipenem (10 μg), meropenem (10 μg), ceftazidime (30 μg), aztreonam (30 μg), nalidixic acid (30 μg), amoxicillin (20/10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), ampicillin (10 μg), amikacin (30 μg), polymixin B (300 units/disc), cefotaxime + clavulanic acid (30/10 μg), ceftriaxone (30 μg) and Piperacillin+tazobactam (100/10 μg) on Mueller-Hinton agar plates by the Kirby Bauer disc diffusion method as per CLSI guidelines (CLSI, 2012 ) and later minimum inhibitory concentration (MIC) by E-strips (HiMedia Laboratories Mumbai, India) was performed for confirmation. Multidrug resistant (MDR) isolates show resistance to ≥3 antibiotic classes (Magiorakos et al., 2012 (link)). Escherichia coli (ATCC) strain 25922 was included as a quality control.
Polymixin b
Manufactured by HiMedia
Sourced in India
Polymixin B is a laboratory-grade antibiotic compound used for research and scientific purposes. It functions as a polypeptide antimicrobial agent effective against a range of Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Ciprofloxacin, by HiMedia (8 mentions)
Amikacin, by HiMedia (7 mentions)
Gentamicin, by HiMedia (6 mentions)
Piperacillin tazobactam, by HiMedia (6 mentions)
Mueller hinton agar (mha), by HiMedia (5 mentions)
Ceftazidime, by HiMedia (5 mentions)
Ampicillin, by HiMedia (4 mentions)
Carbenicillin, by HiMedia (4 mentions)
Imipenem, by HiMedia (4 mentions)
Meropenem, by HiMedia (4 mentions)
Gentamycin, by HiMedia (3 mentions)
Cefotaxime, by HiMedia (2 mentions)
Colistin sulphate, by HiMedia (2 mentions)
Cefepime, by HiMedia (2 mentions)
Levofloxacin, by HiMedia (2 mentions)
Cefoxitin, by HiMedia (2 mentions)
Cotrimoxazole, by HiMedia (2 mentions)
Muller hinton agar, by HiMedia (2 mentions)
Vancomycin, by HiMedia (2 mentions)
E strips, by HiMedia (1 mentions)
9 protocols using polymixin b
1
Antimicrobial Susceptibility Testing of Isolates
2
Antibiotics Susceptibility of KPC-2
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The antibiotic susceptibility was done by Kirby Bauer disc-diffusion method against antibiotics viz. piperacillin-tazobactam (100/10 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), polymixin B (300 units), netilmicin (30 μg) and carbenicillin (100 μg) (Hi-Media, Mumbai, India). Minimum inhibitory concentration was performed by agar dilution method against imipenem, ertapenem, meropenem, cefepime & aztreonam and the results were compared with standard CLSI guidelines [14 ]. The antibiotic susceptibility of the transformants and clone of blaKPC-2 was also determined.
3
Antibiotic Susceptibility Profiling of Bacterial Isolates
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The susceptibility of bacterial isolates against different antibiotics was determined by the disk diffusion method [modified Kirby-Bauer method] on Mueller Hinton agar (Hi-Media, India) following standard procedures recommended by the Clinical and Laboratory Standards Institute (CLSI), Wayne, USA [18 ]. For this purpose following antibiotics with specified concentrations were used; ampicillin (10 μg), ampicillin-sulbactam (10/10 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefepime (30 μg), cefoxitin (30 μg), piperacillin-tazobactam (100/10 μg, aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), gentamycin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), levofloxacin (5 μg), trimethoprim-sulphamethoxazole/co-trimoxazole (25 μg), polymixin B (300unit), colistin sulphate (10 μg)] and tigecycline (30 μg) from HiMedia Laboratories, India. Interpretations of antibiotic susceptibility results were made according to the guidelines of interpretative zone diameters of CLSI [18 ]. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the control organisms for antibiotic sensitivity.
4
Antimicrobial Susceptibility Testing Protocol
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The susceptibility of bacterial isolates against different antibiotics was tested by the disk diffusion method [modified Kirby-Bauer method] on Mueller Hinton Agar (Hi-Media Laboratories, India) following standard zone size interpretative criteria recommended by Clinical and Laboratory Standards Institute (CLSI) [16 ]. Antibiotics that were tested in our study include amoxicillin (AMX 10 μg), amoxicillin clavulanate (AMC 20/10 μg), Gentamycin (GEN 10 μg), Ciprofloxacin (CIP 5 μg), Cotrimoxazole (COT 30 μg), Cefixime (CFM 5 μg), Cefotaxime/Ceftriaxone (CTX/CTR 30 μg), Ceftazidime (CAZ 30 μg), Cefepime (CPM 30 μg), piperacillin tazobactam (PIT 100/10 μg), Imipenem (IMP 10 μg), Meropenem (MEM 10 μg), Polymixin B (PB300 units), and Nitrofurantoin (NI 300 U) (Hi-Media Laboratories, India).
5
Antibiotic Susceptibility Testing Protocol
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Antibiotic susceptibility testing was performed on Mueller-Hinton agar (Himedia, Mumbai, India) plates by Kirby-Bauer disc diffusion method and interpreted as per CLSI recommendations [15 ]. The antibiotics tested viz., ciprofloxacin (5μg), amikacin (30μg), gentamicin (10μg), carbenicillin (10μg), polymixin B (300 μg), ceftazidime (30 μg), Piperacillin-Tazobactam (100/10 μg) (Himedia, Mumbai, India).
Minimum inhibitory concentration (MIC) was determined on Muller Hinton Agar plates by agar dilution method against imipenem and meropenem (Himedia, Mumbai, India) according to CLSI guidelines and interpreted according to CLSI breakpoint [15 ].
Minimum inhibitory concentration (MIC) was determined on Muller Hinton Agar plates by agar dilution method against imipenem and meropenem (Himedia, Mumbai, India) according to CLSI guidelines and interpreted according to CLSI breakpoint [15 ].
6
Antibiotic Susceptibility Testing of Bacterial Isolates
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The susceptibility of bacterial isolates against different antibiotics was determined by the disk diffusion method [modified Kirby-Bauer method] on Mueller Hinton agar (HiMedia, India) following standard procedures recommended by the Clinical and Laboratory Standards Institute (CLSI), Wayne, USA [12 ]. For this purpose, the following antibiotics with specified concentrations were used: ampicillin (10 μg), trimethoprim-sulfamethoxazole/cotrimoxazole (25 μg), gentamycin (10 μg), high level gentamycin (120 μg), amikacin (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cloxacillin (5 μg), erythromycin (15 μg), clindamycin (2 μg), imipenem (10 μg), vancomycin (30 μg), teicoplanin (30 μg), piperacillin-tazobactam (100/10 μg), polymixin B (300 units), and colistin sulphate (10 μg) from HiMedia Laboratories, India. Interpretations of antibiotic susceptibility results were made according to the guidelines of interpretative zone diameters of CLSI [12 ]. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853 were used as the control organisms for antibiotic sensitivity.
7
Antibiotic Susceptibility Testing by Disc Diffusion
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Antibiotic susceptibility testing was performed on Mueller Hinton agar (Himedia, Mumbai, India) plates by Kirby-Bauer disc diffusion method and interpreted as per CLSI recommendations [10 ]. The antibiotics tested viz., meropenem (10μg) ciprofloxacin (5μg), amikacin (30μg), gentamicin (10μg), carbenicillin (10μg), polymixin B (300 μg), ceftazidime (30 μg), Piperacillin-Tazobactam (100/10 μg), faropenem (5μg) (Himedia, Mumbai, India).
Minimum inhibitory concentration (MIC) was determined on Muller Hinton Agar plates by agar dilution method against meropenem and ciprofloxacin (Himedia, Mumbai, India) according to CLSI guidelines and interpreted according to CLSI breakpoint [10 ].
Minimum inhibitory concentration (MIC) was determined on Muller Hinton Agar plates by agar dilution method against meropenem and ciprofloxacin (Himedia, Mumbai, India) according to CLSI guidelines and interpreted according to CLSI breakpoint [10 ].
8
Antibiotic Susceptibility Profiling
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Antibiotic susceptibility test was performed by the Kirby-Bauer disk diffusion test (Baccer et al. 1966) .
Antibiotic discs were placed on freshly prepared lawns of each isolate on Mueller-Hinton agar (MHA) plates and incubated for 24-48 hrs at 37 °C. The diameter of the inhibition zones was measured, and the strains were classi ed following the standard antibiotic disc chart. Standard antibiotic discs were procured from 'HiMedia' which includes Gentamicin (120 µg), Vancomycin (30 µg), Tetracycline (30 µg), Polymixin-B (300 µg), Kanamycin (30 µg), O oxacin (5 µg), Co-Trimoxazole (25 µg), Meropenem (10 µg), Ceftriaxone (30 µg), Clindamycin (2 µg), Ampicillin (10 µg), Nor oxacin (10 µg), Rifampicin (5 µg), Amikacin (30 µg), Penicillin-G 10 µg), Cefdinir (5 µg), Cipro oxacin (5 µg), Azithromycin (15 µg), Methicilin (5 µg), Streptomycin (10 µg).
Antibiotic discs were placed on freshly prepared lawns of each isolate on Mueller-Hinton agar (MHA) plates and incubated for 24-48 hrs at 37 °C. The diameter of the inhibition zones was measured, and the strains were classi ed following the standard antibiotic disc chart. Standard antibiotic discs were procured from 'HiMedia' which includes Gentamicin (120 µg), Vancomycin (30 µg), Tetracycline (30 µg), Polymixin-B (300 µg), Kanamycin (30 µg), O oxacin (5 µg), Co-Trimoxazole (25 µg), Meropenem (10 µg), Ceftriaxone (30 µg), Clindamycin (2 µg), Ampicillin (10 µg), Nor oxacin (10 µg), Rifampicin (5 µg), Amikacin (30 µg), Penicillin-G 10 µg), Cefdinir (5 µg), Cipro oxacin (5 µg), Azithromycin (15 µg), Methicilin (5 µg), Streptomycin (10 µg).
9
Antibiotic Susceptibility Profiling
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The antibiotic susceptibility was done by Kirby Bauer disc diffusion method against antibiotics as piperacillin-tazobactam (100/10 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), polymixin B (300 units) ampicillin (30 μg), cotrimoxazole (10 μg) and carbenicillin (100 μg) (Hi-Media, Mumbai, India). Minimum inhibitory concentration was performed by agar dilution method against imipenem, meropenem, cefepime, aztreonam, cotrimoxazole, ampicillin, & ciprofloxacin and the results were compared with standard CLSI guidelines [10] . The antibiotic susceptibility of the transformants was also determined. Flow diagram of the whole procedure carried out is given in Figure 1.
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