Pure cultures of H. pylori were applied for antibiotic susceptibility test. One strain from each H. pylori-positive sample was selected for this aim. Antimicrobial susceptibility test was accomplished by the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (Merck, Germany) supplemented with 5 % defibrinated sheep blood and 7 % fetal calf serum, according to the Clinical Laboratory Standards Institute (CLSI 2012) [42 ]. The antimicrobial resistance of H. pylori was measured against the widely used antibiotics in cases of H. pylori gastric ulcer. The following antimicrobial disks (HiMedia Laboratories, Mumbai, India) were used: ampicillin (10 μg), metronidazole (5 μg), erythromycin (5 μg), clarithromycin (2 μg), amoxicillin (10 μg), levofloxacin (5 μg), cefsulodin (30 μg), trimethoprim (25 μg), furazolidone (1 μg), moxifloxacin (5 μg), tinidazole (prepared from pure powders, Sigma, 5 μg) and ciprofloxacin (5 μg). After incubation at 37 °C for 48 h in a microaerophilic atmosphere (85 % N2, 10 % CO2 and 5 % O2,), the susceptibility of the H. pylori was measured against each antimicrobial agents. Results were construed in accordance with interpretive criteria provided by CLSI (2012) [42 ]. The H. pylori ATCC 43504 was used a quality control organism in antimicrobial susceptibility determination.
Antimicrobial disks
Manufactured by HiMedia
Sourced in India
Antimicrobial disks are small paper disks impregnated with known concentrations of antimicrobial agents. They are used in the Kirby-Bauer disk diffusion susceptibility test to determine the antimicrobial susceptibility of bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Mueller hinton agar (mha), by Merck Group (3 mentions)
Ampicillin, by HiMedia (2 mentions)
Nutrient broth medium, by HiMedia (2 mentions)
Nutrient agar, by HiMedia (2 mentions)
Tinidazole, by Merck Group (1 mentions)
Metronidazole, by Merck Group (1 mentions)
Amoxicillin, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Amoxicillin, by HiMedia (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Copper 2 sulfate pentahydrate, by Merck Group (1 mentions)
10 protocols using antimicrobial disks
1
Antibiotic Resistance Profiling of H. pylori
2
Antibiotic Susceptibility Profiling of Isolates
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Antibiotic susceptibility analysis was performed, as previously described, by Kirby-Bauer disk diffusion method, on Mueller Hinton agar plates (Qumar et al., 2017 (link)). Antimicrobial disks (Himedia, India) specific for fosfomycin (200 μg), chloramphenicol (30 μg), co-trimoxazole (20 μg), tetracycline (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), doxycycline (30 μg), ciprofloxacin (5 μg), and colistin (10 μg) were used to determine the antibiotic susceptibility profile of the isolates. ESBL production was determined using disk synergy between clavulanic acid and indicator cephalosporins, CTX (cefotaxime) and CAZ (ceftazidime). Both the assays were performed in accordance with Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI, 2013 ). Isolates exhibiting resistance to three or more antimicrobials were designated as multidrug resistant (MDR).
3
Antibiotic Susceptibility of H. pylori
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Pure cultures of H. pylori were applied for antibiotic susceptibility test. One strain from each H. pylori‐positive sample was selected for this aim. Antimicrobial susceptibility test was accomplished by the Kirby–Bauer disk diffusion method using Mueller–Hinton agar (Merck) supplemented with 5% defibrinated sheep blood and 7% fetal calf serum, according to the Clinical Laboratory Standards Institute (CLSI, 2012) 20 . The antimicrobial resistance of H. pylori was measured against the widely used antibiotics in cases of H. pylori gastric ulcer. The following antimicrobial disks (HiMedia Laboratories, Mumbai, India) were used: ampicillin (10 μg), metronidazole (5 μg), erythromycin (5 μg), clarithromycin (2 μg), amoxicillin (10 μg), levofloxacin (5 μg), rifampin (30 μg), cefsulodin (30 μg), trimethoprim (25 μg), furazolidone (1 μg) and spiramycin (100 μg). After incubation at 37 °C for 48 h in a microaerophilic atmosphere (85% N2, 10% CO2, and 5% O2,), the susceptibility of the H. pylori was measured against each antimicrobial agents. Results were construed in accordance with interpretive criteria provided by CLSI 20 . The H. pylori ATCC 43504 was used as quality control organisms in antimicrobial susceptibility determination.
4
Antimicrobial Susceptibility Testing of Bacterial Isolates
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All isolates were also tested for susceptibility to 14 different antimicrobial agents using the disk diffusion method on Mueller Hinton II agar (Bio‐Rad France), following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (EUCAST 2013 ). E. coli ATCC 25922 and ATCC 35218 were used as a control. The antimicrobial disks (Himedia, India) used were nalidixic acid (30 μg), ciprofloxacin (5 μg), ampicillin (10 μg), amoxicillin (25 μg), cefotaxime (30 μg), imipenem (10 μg), tetracycline (30 μg), gentamicin (10 μg), chloramphenicol (30 μg), ceftriaxone (30 μg), norfloxacin (10 μg), ticarcillin (75 μg), amoxicillin/clavulanic acid (30 μg), and sulfamethoxazole/trimethoprim (25 μg). Inhibition diameters of the antibiotics were interpreted according to the European Committee on Antimicrobial Susceptibility Instructions (EUCAST 2013 ). The multiresistance is defined as the resistance to at least three different antibiotics families (Magiorakos et al., 2011 ).
5
Antibiotic Resistance Profiling of Salmonella
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The antimicrobial susceptibility test for all the Salmonella isolates was performed and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines, using disk diffusion assay. Escherichia coli ATCC 25922 was used as a quality control strain [13 ]. A total of nine antimicrobial disks (HiMedia, India) comprising ampicillin (10 μg), ceftriaxone (30 μg), tetracycline (30 μg), ciprofloxacin (10 μg), co-trimoxazole (25 μg), imipenem (10 μg), nalidixic acid (30 μg), sulfisoxazole (50 μg), and chloramphenicol (30 μg) were tested. The zone of inhibition (diameter in mm) for each antibiotic was initially measured for the quality control strain, followed by all the test strains (Supplement Table-1). The antimicrobial susceptibility assay was performed in triplicate and the data obtained were compared with CLSI interpretative chart.
6
Antimicrobial Evaluation of Copper Sulfate and DMSO
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Copper (II) sulfate pentahydrate (CuSO4.5H2O) and Dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Nutrient agar, nutrient broth medium and antimicrobial disks were purchased from Hi-media Labs, Mumbai, India. The clinical isolates of bacterial and fungal cultures were kindly provided by Microbiology Lab, Department of Ilmul Advia and Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh and R2B strain of NDV from Indian Veterinary Research Institute, Izzatnagar, Bareilly (U.P). All bacterial strains were resistant to Cefixime, Amoxyclav, Cefotaxime, Methicillin and Ampicillin antibiotics.
7
Antibiotic Susceptibility of H. pylori
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Pure cultures of H. pylori were applied for antibiotic susceptibility test. One strain from each H. pylori-positive sample was selected for this aim. Antimicrobial susceptibility test was accomplished by the Kirby-Bauer disc diffusion method using Mueller–Hinton agar (Merck, Germany) supplemented with 5 % defibrinated sheep blood and 7 % fetal calf serum, according to the Clinical Laboratory Standards Institute [15 ]. The antimicrobial resistance of H. pylori was measured against the widely used antibiotics in cases of H. pylori gastric ulcer. The following antimicrobial disks (HiMedia Laboratories, Mumbai, India) were used: ampicillin (10 µg), metronidazole (5 µg), erythromycin (5 µg), clarithromycin (2 µg), amoxicillin (10 µg), tetracycline (30 µg), levofloxacin (5 µg), streptomycin (10 µg), rifampin (30 µg), cefsulodin (30 µg), trimethoprim (25 µg), furazolidone (1 µg) and spiramycin (100 µg). After incubation at 37 °C for 48 h in a microaerophilic atmosphere, the susceptibility of the H. pylori was measured against each antimicrobial agents. Results were construed in accordance with interpretive criteria provided by CLSI (2012) [15 ]. The H. pylori ATCC 43504 was used as quality control organisms in antimicrobial susceptibility determination.
8
Gonococcal Antimicrobial Susceptibility Profiling
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Two thin sterile cotton-tipped urethral swabs were collected for Gram stain and direct inoculation onto a plate of Modified Thayer Martin medium (MTM). The plates were transported to the laboratory inside a screw-capped, wide-mouth plastic container with a candle lit inside (3–7% CO2). Growth of N. gonorrhoeae was suspected on the appearance of small pin-point, grey-to-white, smooth, translucent, raised convex colonies of Gram-negative cocci, oxidase-positive, superoxol-positive, and rapid carbohydrate utilisation test (RCUT), showing glucose fermentation-positive and maltose-, lactose-, and sucrose-negative. All gonococcal isolates were subjected to anti-microbial susceptibility testing by the disk diffusion method as per Clinical and Laboratory Standards guidelines (2011) using anti-microbial disks (HIMEDIA Laboratories Pvt. Ltd., Mumbai, India).[8 ] Control strains used were provided by Apex Regional STI Centre, Safdarjang Hospital, New Delhi. All gonococcal isolates were tested for production of ß-lactamase by the chromogenic cephalosporin method (Nitrocefin disc). The minimum inhibitory concentration (MIC) of ceftriaxone for N. gonorrhoeae isolates was determined by the E test method using Ceftriaxone Ezy MIC™ Strip (CTR) (0.016–256 µg/ml) (HIMEDIA Catalogue Number: EM066).
9
Antimicrobial Evaluation of Herbal Extracts
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All solvents used were of HPLC grade obtained from Sigma-Aldrich. Nutrient agar, Nutrient broth medium, and antimicrobial disks were purchased from Hi-media Labs, Mumbai. The bacterial cultures were kindly provided by Microbiology Lab, Department of Ilmul Advia and Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh.
10
Antimicrobial Disk Diffusion for ESBL Detection
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The antimicrobial disks (HI Media, Mumbai, India) used were CTX (30 µg), CAZ (30 µg), cefotaxime-clavulanic acid (CEC) (30 µg/10 µg) and ceftazidimeclavulanic acid (CAC) (30 µg/10 µg). Each disk was kept at least 20 mm apart, center to center. Isolates resistant to CTX and CAZ but sensitive to CEC and CAC with enhanced ZOI ≥ 5 mm was con rmed as ESBL producers [6].
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