Antimicrobial sensitivity testing was done by Kirby-Bauer Antimicrobial Susceptibility Test (disc diffusion method) using Mueller-Hinton agar [15 ]. Six different antibiotics were tested and the zone size was measured. Amikacin, streptomycin, gentamicin, tobramycin, netilmicin, and neomycin (obtained from HiMedia, India) were taken and screening test for detection of high level aminoglycoside resistance (HLAR) in Enterococcus species was confirmed as per Clinical and Laboratory Standards Institute (CLSI) approved standards [16 ] The E. coli isolates were described as isolates with high level aminoglycoside resistance considering growth ≥2048 μg/mL for streptomycin, ≥512 μg/mL for gentamicin, ≥512 μg/mL for neomycin, ≥256 μg/mL for tobramycin, ≥256 μg/mL for netilmicin, and ≥256 μg/mL for Amikacin. MICs were determined by Etest (AB Biodisk).
Tobramycin
Manufactured by HiMedia
Sourced in India
Tobramycin is a broad-spectrum aminoglycoside antibiotic used in laboratory settings. It is effective against a variety of gram-negative bacteria. Tobramycin functions by inhibiting bacterial protein synthesis, leading to cell death.
Automatically generated - may contain errors
Lab products found in correlation
Amikacin, by HiMedia (17 mentions)
Gentamicin, by HiMedia (14 mentions)
Ciprofloxacin, by HiMedia (13 mentions)
Ampicillin, by HiMedia (10 mentions)
Ceftazidime, by HiMedia (10 mentions)
Imipenem, by HiMedia (10 mentions)
Cefotaxime, by HiMedia (9 mentions)
Ceftriaxone, by HiMedia (7 mentions)
Meropenem, by HiMedia (7 mentions)
Colistin, by HiMedia (7 mentions)
Aztreonam, by HiMedia (7 mentions)
Levofloxacin, by HiMedia (6 mentions)
Cefepime, by HiMedia (6 mentions)
Netilmicin, by HiMedia (5 mentions)
Piperacillin, by HiMedia (5 mentions)
Tetracycline, by HiMedia (5 mentions)
Cotrimoxazole, by HiMedia (5 mentions)
Norfloxacin, by HiMedia (5 mentions)
Nalidixic acid, by HiMedia (5 mentions)
Streptomycin, by HiMedia (4 mentions)
19 protocols using tobramycin
1
Antimicrobial Resistance Profiling in Enterococcus
2
Disk Diffusion Antibiotic Susceptibility
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Disk diffusion test was performed in accordance with the Clinical Laboratory Standard Institute (CLSI, 2016).
9
The AGs tested were amikacin (30 µg), gentamicin (10 µg), tobramycin (10 µg), and netilmicin (10 µg) (HiMedia Laboratories).
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Disk diffusion test was performed in accordance with the Clinical Laboratory Standard Institute (CLSI, 2016).
9
The AGs tested were amikacin (30 µg), gentamicin (10 µg), tobramycin (10 µg), and netilmicin (10 µg) (HiMedia Laboratories).
3
Antibiotic and Quercetin MIC Determination
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For establishing the MIC, the following antibiotics were used; amikacin, meropenem, levofloxacin, chloramphenicol, gentamycin, tobramycin, ceftriaxone and piperacillin (HiMedia, India). Quercetin (Cat No: Q4951) purchased from Sigma Aldrich (USA) was used.
The MIC of antibiotics and Quercetin against the strains were tested by broth micro-dilution method using 96-well plates, using Muller Hinton broth (MHB) (HiMedia, India) according to Clinical & Laboratory Standards Institute (CLSI) guidelines for MIC breakpoints [18 (link)]. Briefly, the antibiotics stocks (10 mg/mL) were prepared using sterile distilled water and Quercetin stock was prepared in dimethyl sulfoxide (DMSO) (10 mg/mL). Fresh MHB containing antibiotics (amikacin, tobramycin, levofloxacin, gentamycin and ceftriaxone) in a broad range between 0.5 to 128 μg/mL was inoculated with overnight cultures of the bacteria at a cell density of 1×105 CFU/mL and incubated at 37°C for 24 h. The antibiotics showing difference of more than 4 μg/mL between visible growth and no visible growth, were further tested in a narrow range between the two concentrations with an increment of 1 μg/mL to establish the exact MIC value. After incubation, the plates were observed for visible growth and MIC was interpreted as the lowest concentration of the antibiotic at which no visible growth was observed.
The MIC of antibiotics and Quercetin against the strains were tested by broth micro-dilution method using 96-well plates, using Muller Hinton broth (MHB) (HiMedia, India) according to Clinical & Laboratory Standards Institute (CLSI) guidelines for MIC breakpoints [18 (link)]. Briefly, the antibiotics stocks (10 mg/mL) were prepared using sterile distilled water and Quercetin stock was prepared in dimethyl sulfoxide (DMSO) (10 mg/mL). Fresh MHB containing antibiotics (amikacin, tobramycin, levofloxacin, gentamycin and ceftriaxone) in a broad range between 0.5 to 128 μg/mL was inoculated with overnight cultures of the bacteria at a cell density of 1×105 CFU/mL and incubated at 37°C for 24 h. The antibiotics showing difference of more than 4 μg/mL between visible growth and no visible growth, were further tested in a narrow range between the two concentrations with an increment of 1 μg/mL to establish the exact MIC value. After incubation, the plates were observed for visible growth and MIC was interpreted as the lowest concentration of the antibiotic at which no visible growth was observed.
4
Antibiotic Susceptibility of Bacterial Isolates
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The susceptibility to the commercial antibiotics of the bacterial isolates was evaluated using the disc diffusion method. Antibiotics used against Gram-positive bacteria included cefoxitin, benzyl-penicillin, oxacillin, imipenem, gentamicin, ciprofloxacin, moxifloxacin, inducible clindamycin resistance, erythromycin, clindamycin, vancomycin, tetracycline, fusidic acid, and trimethoprim/sulfamethoxazole. On the other hand, antibiotics used against Gram-negative bacteria included temocillin, ampicillin, amoxicillin/clavulanic acid, ticarcillin, ticarcillin/clavulanic acid, piperacillin, piperacillin/tazobactam, cephalothin, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefepime, ertapenem, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, tigecycline, fosfomycin, nitrofurantoin, pefloxacin, minocycline, colistin, and trimethoprim/sulfamethoxazole (Himedia Labs, Mumbai, India) [19 ].
5
Antimicrobial Susceptibility Profiling
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A total of 18 clinically relevant antibiotics were tested using the disc diffusion method (Kirby-Bauer's) and the inhibition zone diameters were measured by (mm) according to Clinical and Laboratory Standards Institute (CLSI) guidelines M100 27 th 18 . These antimicrobial discs were amoxicillin/clavulanic acid ( 30μg), ampicillin (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), ertapenem(10 μg), gentamicin (10 μg), tobramycin (10 μg) , amikacin (30 μg), tetracycline (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), nalidixic acid (30 μg), co-trimoxazole (25 μg) and colistin (10 μg) were obtained from HiMedia Laboratories (India). The results for the antimicrobial susceptibility test strain were interpreted as (S) susceptible, (I) intermediate, or (R) resistant by comparing the results to the CLSI 2017 18 standard zone diameter Quality control strains used in antimicrobial susceptibility testing are Escherichia coli ATCC #25922 18 . Minimum inhibitory concentration (MIC) of imipenem and meropenem were determined using the agar dilution method and interpreted according to the CLSI guidelines, the carbapenems resistant Enterobacteriaceae (CRE) isolates were included based on showing MICs ≥2 µg/mL for imipenem or meropenem were considered resistant. Only one isolate per patient was included 19 .
6
Antimicrobial Susceptibility Testing of Escherichia coli
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This was performed with the disk diffusion method (Kirby-Bauer’s) and the inhibition zone diameters were measured by (mm) according to Clinical and Laboratory Standards Institute (CLSI).12 (link) The antimicrobial discs tested include Amoxicillin/clavulanic acid (30 μg), ampicillin (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), gentamicin (10 μg), tobramycin (10 μg), amikacin (30 μg), tetracycline (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), nalidixic acid (30 μg), co-trimoxazole (25 μg) and colistin (10 μg) were obtained from HiMedia Laboratories (India). The results for the antimicrobial susceptibility test strain were interpreted as (S) susceptible, (I) intermediate or (R) resistant by comparing the results to the CLSI 2018 standard zone diameter.12 (link) Minimum inhibitory concentrations (MICs) for imipenem and meropenem were performed by the agar dilution method and interpreted according to CLSI guidelines (strains displaying MICs ≥8 µg/mL for imipenem and meropenem were considered resistant).12 (link)
Escherichia coli ATCC 25922 was used as standard control strains.
Escherichia coli ATCC 25922 was used as standard control strains.
7
Enzymatic Assay for Cysteine Sulfoxide Synthesis
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Reduced
form of nicotinamide adenine dinucleotide
(NADH), lactate dehydrogenase (LDH) from rabbit muscle, and (±)-L-alliin
were purchased from Sigma-Aldrich; pyridoxal 5′-phosphate (PLP)
andd ,l -dithiothreitol (DTT) were from Serva; kanamycin
is a domestic product (OAO Biokhimik); DEAE-sepharose was from Amersham.
2-Nitro-5-thiobenzoate (NTB) was prepared according to ref (31 (link)). S-Methyl-l -cysteine sulfoxide (methiin) was synthesized according to
Morozova et al.22 (link) PEG–poly(α,β-aspartic
acid)70 (PEG–P(Asp)70) and poly-(l -lysine)70 (PLL70) were synthesized according
to Koide et al.32 (link) Luria–Bertani
broth (LB), Mueller–Hinton broth, Mueller–Hinton agar,
and antibiotic-impregnated discs: amikacin, amoxycillin/clavulanic
acid, ampicillin, azithromycin, aztreonam, cefepime, ceftazidime,
ceftriaxone, cephotaxime, cephoxitin, chloramphenicol, ciprofloxacin,
colistin, doxycycline, erythromycin, gentamicin, imipenem, levofloxacin,
lincomycin, norfloxacin, ofloxacin, oxacillin, rifampicin, spiramycin,
tobramycin, and vancomycin were from HiMedia Laboratories Pvt. Limited
(India).
form of nicotinamide adenine dinucleotide
(NADH), lactate dehydrogenase (LDH) from rabbit muscle, and (±)-L-alliin
were purchased from Sigma-Aldrich; pyridoxal 5′-phosphate (PLP)
and
is a domestic product (OAO Biokhimik); DEAE-sepharose was from Amersham.
2-Nitro-5-thiobenzoate (NTB) was prepared according to ref (31 (link)). S-Methyl-
Morozova et al.22 (link) PEG–poly(α,β-aspartic
acid)70 (PEG–P(Asp)70) and poly-(
to Koide et al.32 (link) Luria–Bertani
broth (LB), Mueller–Hinton broth, Mueller–Hinton agar,
and antibiotic-impregnated discs: amikacin, amoxycillin/clavulanic
acid, ampicillin, azithromycin, aztreonam, cefepime, ceftazidime,
ceftriaxone, cephotaxime, cephoxitin, chloramphenicol, ciprofloxacin,
colistin, doxycycline, erythromycin, gentamicin, imipenem, levofloxacin,
lincomycin, norfloxacin, ofloxacin, oxacillin, rifampicin, spiramycin,
tobramycin, and vancomycin were from HiMedia Laboratories Pvt. Limited
(India).
8
Antibiotic Susceptibility Testing Protocol
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Minimum inhibitory concentration (MICs) of the study isolates against gentamicin, kanamycin, amikacin, streptomycin, tobramycin, and netilmicin (Hi-Media, India) were determined by agar dilution method. For determination of susceptibility against other groups of antimicrobials, Kirby Baur disc diffusion method was used. Antibiotics tested were imipenem (10 µg), cefepime (30 µg), aztreonam (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), and streptomycin (10 µg) (Hi-Media, India). Results were interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines 2017 [5 (link)]. Escherichia coli
9
Antimicrobial Resistance Profiling of ACBL E. coli
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All the ACBL producing E. coli isolates were subjected for their sensitivity and resistance to different antimicrobials by disc diffusion method (CLSI 2014). The antibiotics used were cotrimoxazole (25 µg), cefoperazone (75 µg), chloramphenicol (30 µg), ampicillin (10 µg), cefoxitin (30 µg), gentamicin (10 µg), tetracycline (30 µg), amikacin (30 µg), levofloxacin (5 µg), penicillin-G (10 U), tobramycin (30 µg), ceftriaxone (30 µg), ceftizoxime (30 µg), ceftazidime (30 µg) and cefotaxime (30 µg) (HiMedia, India).
Detection of PMQR (qnrA, qnrB, qnrS) and sulfonamide resistance genes (sul1, sul2, sul3)
All the levofloxacin and co-trimoxazole resistant ACBL-producing E. coli isolates were studied for detection of plasmid mediated quinolone resistance genes and sul1, sul2, and sul3 genes, respectively (Frank et al. 2007; Kar et al. 2015) .
Detection of PMQR (qnrA, qnrB, qnrS) and sulfonamide resistance genes (sul1, sul2, sul3)
All the levofloxacin and co-trimoxazole resistant ACBL-producing E. coli isolates were studied for detection of plasmid mediated quinolone resistance genes and sul1, sul2, and sul3 genes, respectively (Frank et al. 2007; Kar et al. 2015) .
10
Antibiotic Susceptibility Profiling of A. hydrophila
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Antibiotic susceptibility profile for A. hydrophila Ah17 was determined by the Kirby‐Bauer disk diffusion method (Bauer, Kirby, Sherris, & Turck, 1966 ). The following antibiotics were tested: Amikacin (AK: 30 μg), Amoxicillin (AMC: 30 μg), Ampicillin (AMP: 10 μg), Azithromycin (AZM: 15 μg), Cefixime (CFM: 5 μg), Cefoperazone (CPZ: 75 μg), Cefpodoxime (CPD: 10 μg), Ciprofloxacin (CIP: 5 μg), Chlorompenicol (C: 30 μg), Clarithromycin (CLR: 15 μg), Co‐Trimoxazole (COT: 25 μg), Doxycycline hydrochloride (DO: 30 μg), Fosfomycin (FO: 200 μg), Fusidic acid (FC: 10 μg), Gentamicin (GEN: 10 μg), Imipenem (IPM: 10 μg), Kanamycin (K: 30 μg), Linezolid (LZ: 30 μg), Methicillin (MET: 5 μg), Minocycline (MI: 30 μg), Nalidixic acid (NA: 30 μg), Nitrofurantoin (NIT: 300 μg), Norfloxacin (NX: 10 μg), Penicillin G (P: 10 μg), Pristinomycin (RP: 15 μg), Rifampicin (RIF: 5 μg), Streptomycin (S: 10 μg), Teicoplanin (TEI: 30 μg), Tetracycline (TE: 30 μg), Trimethoprim (TR: 5 μg), Tobramycin (TOB: 10 μg), and Vancomycin (VA: 30 μg) (HiMedia). 200 μl of A. hydrophila Ah17 suspension was swabbed on Mueller‐Hinton agar (MHA) medium (HiMedia) and kept for drying. Antibiotic discs were placed on the MHA medium and incubated at 37°C for 24–48 hr. The diameter of the zone of inhibition was measured, and susceptibility was categorized according to the manufacturer's protocol.
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