Two clinical isolates of K. pneumoniae sourced from the Microbiology and Immunology Department stock culture collection at the Faculty of Pharmacy, Zagazig University, were used. The clinical isolates were characterized using 16S rRNA gene sequencing, and the obtained results were deposited in GenBank (https://www.ncbi.nlm.nih.gov/ ) under accession numbers ON798797 and ON798801 (Abdel-Halim et al., 2022 (link)). Pantoprazole was supplied by Novartis, Egypt. Meropenem was supplied by AstraZeneca, Egypt. Captopril and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, United States). Antibiotic disks and bacterial culture media were obtained from Oxoid, United Kingdom.
Antibiotic disk
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Antibiotic disks are small, circular paper disks impregnated with a specific antibiotic. They are used in the Kirby-Bauer disk diffusion susceptibility test, a standard method for determining the antibiotic susceptibility of bacterial isolates.
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Lab products found in correlation
Etest strips, by bioMérieux (4 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (4 mentions)
Etest, by bioMérieux (3 mentions)
Vitek 2 system, by bioMérieux (2 mentions)
Imipenem, by Thermo Fisher Scientific (2 mentions)
Ampicillin, by Thermo Fisher Scientific (2 mentions)
Mueller hilton agar, by HiMedia (2 mentions)
Sodium chloride (nacl), by Vivantis Technologies (2 mentions)
Api 20e, by bioMérieux (2 mentions)
Cefoxitin, by Thermo Fisher Scientific (2 mentions)
Nalidixic acid, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Ampicillin sulbactam, by Thermo Fisher Scientific (2 mentions)
Mh agar, by Thermo Fisher Scientific (2 mentions)
Mueller hinton agar (mha), by Merck Group (2 mentions)
Bacterial culture media, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Captopril, by Merck Group (1 mentions)
Meropenem, by AstraZeneca (1 mentions)
Colistin sulfate, by Chem-Impex (1 mentions)
88 protocols using antibiotic disk
1
Characterization of K. pneumoniae Isolates
2
Peptide Synthesis and Antibiotic Acquisition
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K11 with C-terminal amidation was synthesized by GenScript (Piscataway, NJ, USA) using solid-phase Fmoc chemistry and purified to >90% purity using HPLC. The mass was confirmed by MALDI-TOF mass spectroscopy. The antibiotics were acquired from TCI (Tokyo, Japan), except colistin sulfate, which was obtained from Chem-Impex Int’l Inc. (Wood Dale, IL, USA). Antibiotic disks were purchased from Oxoid (Oxoid Ltd., UK).
3
Antibiotic Susceptibility Profiling of P. aeruginosa
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Overnight cultures of P. aeruginosa PAO1161 and mutant strains were diluted 1:100 in 15 ml of fresh L‐broth medium and incubated at 37°C to OD600 equal to 0.1. Bacteria were spread evenly on Mueller–Hinton plates (Biomaxima S.A; 17.6 g/L casein hydrolase, 2.0 g/L beef extract, 1.5 g/L starch, 17 g/L agar) to give a homogenous lawn, and then, antibiotic disks (Oxoid) were placed on the center of each plate. Disks with antibiotics from different groups such as cephalosporins: ceftazidime (10 µg) (CAZ); quinolones: ciprofloxacin (5 µg) (CIP); β‐lactams: imipenem (10 µg) (IPM), meropenem (10 µg) (MEM), and piperacillin (100 µg) (PRL); polymyxins: polymyxin B (300 µg) (PB) and colistin (10 µg) (CT); and aminoglycosides: tobramycin (10 µg) (TOB) were used. The plates were incubated at 37°C for 20 hr, and the diameter of growth inhibition was measured. The tests were repeated several times.
4
Antibiotic Susceptibility Testing Protocol
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Antibiotic susceptibility disk diffusion testing was conducted according to CLSI guidelines (20 ) using the following antibiotic disks from Oxoid (Nepean, Ontario, Canada): ampicillin 10 µg; chloramphenicol 30 µg; ceftriaxone 30 µg; trimethoprim–sulfamethoxazole 25 µg; amoxicillin–clavulanic acid 30 µg; cefaclor 30 µg; ciprofloxacin 5 µg; moxifloxacin 5 µg; clarithromycin 15 µg; azithromycin 15 µg; tetracycline 30 µg; levofloxacin 5 µg and meropenem 10 µg. Production of β-lactamase was determined using DrySlide Nitrocefin (Becton Dickinson).
5
Antimicrobial Susceptibility Testing Protocol
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The disk diffusion technique was used to determine antimicrobial sensitivity and resistance after isolating colonies and growing them on Mueller-Hinton agar (Oxoid) at 37°C for 24 h. Following antibiotic disks (Oxoid) were used with the given concentrations: ampicillin (10 μg), chloramphenicol (30 μg), streptomycin (10 μg), sulfonamide (300 μg), trimethoprim (5 μg), ciprofloxacin (5 μg), tetracycline (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), Cefotaxime (5 μg), Mecillinam (10 μg) and Imipenem (10 μg). The criteria published by the Clinical and Laboratory Standards Institute (CLSI) was used for the interpretation of the results.
6
Antimicrobial Susceptibility Testing of Bacteria
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The antimicrobial susceptibility test of the isolated bacterium was performed using 21 varieties of antibiotic disks (Oxoid, Hampshire, UK) that are recommended by the Clinical and Laboratory Standard Institute (CLSI) guideline. The list of antibiotics is summarized in Table 1 . Standard disk diffusion method was conducted on Muller Hinton Agar (BD Difco, New Jersey, USA) at 27 °C for 24 h. Susceptibility and resistance were also determined according to the CLSI guideline [33 ]. Escherichia coli (ATCC 25922) was used in the experiment to clarify the strain criteria. For the biochemical analysis, VITEKⓇ2 System (bioMérieuxⓇ, Marcy-l’Étoile, France) was performed using a gram-negative colorimetric identification card following manufacturer’s protocols. V. coralliilyticus 58, which is reported to be a strain with high virulence to Pacific oyster larvae [7 (link),27 (link)], was also analyzed using the anti-microbial susceptibility test and biochemical test to compare with the isolated bacterium.
7
Antimicrobial Susceptibility Testing Protocols
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Antimicrobial susceptibility testing was done on Mueller-Hinton II agar (MH-II agar) (Becton Dickinson, Heidelberg, Germany) for all strains except for C. tuberculostearicum CIP102622 which was grown on a modified MH-II agar completed with 1% Tween 80. The disk diffusion technique was used according to the EUCAST (Société Française de Microbiologie, 2019 ). All tested antibiotic disks (Oxoid, Basingstoke, Hampshire, United Kingdom) are listed in Supplementary Table 2 . Antibiotics susceptibility profiles were interpreted on the basis of the EUCAST guide (Société Française de Microbiologie, 2019 ).
8
Antibiotic Susceptibility Testing of Klebsiella
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Antimicrobial susceptibility testing was performed by the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) (2020) . All antibiotics recommended for Enterobacterales were tested, considering the particularities for Klebsiella spp. For this approach 37 different antibiotic disks (Oxoid Ltd., Basingstoke, United Kingdom) were used (Supplementary Material 1 ). Each strain was considered susceptible or non-susceptible (either intermediate or resistant) to each antibiotic tested. Based on the susceptibility profile, each strain was classified into different categories including, multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) (Magiorakos et al., 2012 (link)).
9
Antibiotic Susceptibility Testing of Salmonella Typhi
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Dilution was carried out from bacterial suspensions obtained from Salmonella Typhi cultures to determine the turbidity level using McFarland 0.5 turbidity standards containing 1.5 × 108/mL bacteria. Bacteria were then plated on the surface by swabbing them evenly on Müller-Hinton agar medium (Oxoid), then left for 10 minutes so that the bacteria could stick to the surface of the media. Each disc containing antibiotics was then placed on Müller-Hinton agar and incubated at 37°C for 24 hours. The antibiotic disks (Oxoid) contained ampicillin (10 μg), amoxicillin (30 μg), sulfamethoxazole/trimethoprim (SXT; 25 μg), ceftriaxone (30 μg), cefepime (30 μg), cefixime (5 μg), ofloxacin (5 μg) and chloramphenicol (30 μg). The diameter of the inhibition zone was measured and interpreted on the basis of Clinical and Laboratory Standards Institute criteria. Specifically, organisms were considered resistant if the diameter of the zone of inhibition was ≤13 mm for ampicillin, 13 mm for amoxicillin, 10 mm for SXT, 13 mm for ceftriaxone, 18 mm for cefepime, 15 mm for cefixime, 12 mm for ofloxacin and 12 mm for chloramphenicol [18 ].
10
Antimicrobial Susceptibility Testing of Isolates
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Antimicrobial susceptibility tests were performed using the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2018 ). The antibiotic disks (Oxoid, UK) ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg), piperacillin-tazobactam (100/10 µg), cefoperazone-sulbactam (75/30 µg), cefazolin (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), gentamicin (10 µg), amikacin (30 µg), netilmicin (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), norfloxacin (10 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), and fosfomycin (200 µg) were used. Escherichia coli ATCC 25922 was used as a control in all antibiogram tests. Whether a strain was MDR was determined on the basis of acquired non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012 (link)).
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