Antimicrobial resistance profiling was performed using the most representative antimicrobial agents from the different antibiotic families, which are of great clinical and epidemiological relevance. Kirby-Bauer disk diffusion or broth microdilution methods (in the case of colistin) were done according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020 ). Escherichia coli ATCC 25922 was used as a control strain. The antimicrobials tested by disk diffusion were: amoxicillin/clavulanate (20/10 μg), ampicillin (10 μg), aztreonam (30 μg), cefepime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), fosfomycin (200 μg/50 μg of glucose-6-phosphate), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg) (Becton Dickinson). Extended-spectrum beta-lactamases were screened by the ESBL test following the CLSI guidelines (CLSI, 2020 ). Isolates were classified as susceptible, resistant to 1 or 2 antimicrobial categories, multidrug-resistant (MDR) if resistant to at least one agent in ≥ 3 antimicrobial categories; or extensively drug-resistant (XDR), if resistant to at least one agent in all but two or fewer antimicrobial categories (Magiorakos et al., 2012 (link)).
Gentamicin
Manufactured by BD
Sourced in United States, Germany, Switzerland
Gentamicin is a broad-spectrum antibiotic used in laboratory settings. It is an aminoglycoside antibiotic that inhibits bacterial protein synthesis, thereby preventing bacterial growth and replication. Gentamicin is commonly used in cell culture media to prevent bacterial contamination.
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Lab products found in correlation
Ciprofloxacin, by BD (42 mentions)
Tetracycline, by BD (35 mentions)
Chloramphenicol, by BD (28 mentions)
Cefotaxime, by BD (25 mentions)
Trimethoprim sulfamethoxazole, by BD (24 mentions)
Ampicillin, by BD (22 mentions)
Amikacin, by BD (19 mentions)
Nalidixic acid, by BD (17 mentions)
Ceftazidime, by BD (17 mentions)
Meropenem, by BD (16 mentions)
Kanamycin, by BD (14 mentions)
Streptomycin, by BD (14 mentions)
Cefepime, by BD (14 mentions)
Cefoxitin, by BD (14 mentions)
Imipenem, by BD (13 mentions)
Erythromycin, by BD (13 mentions)
Clindamycin, by BD (11 mentions)
Amoxicillin clavulanic acid, by BD (10 mentions)
Penicillin, by BD (9 mentions)
Tobramycin, by BD (8 mentions)
67 protocols using gentamicin
1
Antimicrobial Resistance Profiling of Bacteria
2
Antimicrobial Susceptibility Testing
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Bacterial cell suspensions in saline were normalized at 0.5 McFarland standard and swabbed onto MH agar plates. Disks containing ciprofloxacin (5 μg), imipenem (10 μg), gentamicin (10 μg), novobiocin (30 μg), erythromycin (15 μg), rifampicin (5 μg), or tobramycin (10 μg) (Becton Dickinson) were placed on the surface of the inoculated plates, and growth inhibition halo diameters were measured after 24-h incubation at 37°C.
3
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility testing was performed using the disk-diffusion method and the antibiotics ampicillin (AM), amoxicillin-clavulanic acid (AMC), cefotaxime (CTX), nalidixic acid (Na), ciprofloxacin (CIP), gentamicin (GM), kanamycin (K), streptomycin (S), sulfamethoxazole (Su), trimethoprim (TMP), tetracycline (T), and chloramphenicol (C) (Becton Dickinson, Heidelberg, Germany). Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) performance standards (CLSI, 2016 ). For sulfamethoxazole, for which breakpoints are not listed separately from trimethoprim, an inhibition zone of ≤10 mm was interpreted as resistant. Isolates displaying resistance to three or more classes of antimicrobials (counting β-lactams as one class) were defined as multidrug-resistant (MDR). Synergistic effects between AMC and CTX were regarded as an indication of the presence of an ESBL producer (Kaur et al., 2013 (link)).
4
Antimicrobial Susceptibility Analysis
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A detailed antimicrobial (IMP, meropenem, cefoxitin, ceftazidime, cefpodoxime, ceftriaxone, amikacin, gentamicin, trimethoprim–sulfamethoxazole, polymyxin B, colistin, tigecycline, and fluoroquinolones (Becton Dickinson, Sparks, MD, USA) susceptibility analysis was conducted using the disc diffusion method according to CLSI guidelines.
5
Antibiotic Susceptibility Profiling of Carbapenem-Resistant Isolates
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Determination of the antibiotic susceptibility profile of the 40 selected carbapenem‐resistant isolates was performed using the CLSI disk diffusion method (Clinical & Laboratory Standards Institute, 2018 ) and the reference strain Escherichia coli ATCC 25922 as quality control, as previously described (Harmon et al., 2019 ). The meropenem, imipenem, cefotaxime, ciprofloxacin, gentamicin, and tetracycline antibiotic disks were purchased from Becton Dickinson. To determine whether an isolate was susceptible, intermediate, or resistant to an antibiotic, we used CLSI zone diameter breakpoint values (Clinical & Laboratory Standards Institute, 2018 ). Unless otherwise indicated, for taxa in which the CLSI zone diameter breakpoints are not provided, we used the CLSI Enterobacteriaceae breakpoint values (Clinical & Laboratory Standards Institute, 2018 ).
6
Isolation of Aortic Endothelial Cells
7
Antimicrobial Susceptibility Testing of Isolates
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Antimicrobial susceptibility testing (AST) was conducted on the isolates using the disk-diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [11 ]. Antimicrobial substances included ampicillin, amoxicillin/clavulanic acid, cefazolin, cefotaxime, cefepime, nalidixic acid, ciprofloxacin, sulfamethoxazole-trimethoprim, fosfomycin, azithromycin, nitrofurantoin, streptomycin, kanamycin, gentamicin, chloramphenicol, and tetracycline (Becton, Dickinson, Heidelberg, Germany). Results were interpreted according to CLSI breakpoints for human clinical isolates [11 ]. Multidrug resistance (MDR) was defined as resistance to three or more classes of antimicrobials, counting beta-lactams as one class.
8
Antibiotic Susceptibility Testing Protocol
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Antimicrobial activity of all antibiotic solutions was measured by the Kirby-Bauer disk diffusion method, performed following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol.19 (link),20 Reference strains E. coli NCTC 12241, P. aeruginosa PAO1, or S. aureus NCTC 12973, were chosen depending on the spectrum of activity of the antibiotic tested, and are shown in Table I . Samples were tested in triplicate using 7 mm paper disks (Becton Dickinson, Heidelberg, Germany), impregnated with 20 µl sample solution. The disks were placed on circular Petri dishes with 25 ml Mueller Hinton agar (Thermo Fisher Scientific Oxoid Ltd, Basingstoke, United Kingdom), which was inoculated with a suspension of the reference organism. Each test batch included a control disk containing 10 µg gentamicin (Becton Dickinson) to ascertain proper preparation and application of the bacterial suspension. Plates were incubated at 37°C for 20 hours, at which time zones of inhibition (ZOI) were measured to the nearest whole millimetre.
9
Antibiotic Resistance Screening in Environmental Isolates
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Samples were first plated on Chromocult agar, and then presumptive E. coli colonies were transferred onto MacConkey lactose (MKL) agar for confirmation before replating on Chromocult was performed to ensure pure isolates. We selected up to four E. coli colonies from soil and fecal samples and two from water and surface samples to test for AR. More isolates from soil samples than from water or surfaces were used because we expected higher microbial diversity in soil. Antibiotic sensitivity was assessed using the Kirby-Bauer disc diffusion method (43 (link)) and 12 antibiotics: ampicillin, amoxicillin/clavulanate, cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, enrofloxacin, gentamicin, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline (Becton, Dickinson, Franklin Lakes, NJ). Zones of inhibition were measured after a 24-h incubation period using digital calipers.
10
Antibiotic Susceptibility Testing for Gram-Negative Isolates
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Determination of the antibiotic susceptibility profile for carbapenems and other antibiotics (Table 2 ; Table 3 ; and Figure 2 ) for each selected isolate was performed using the disk diffusion method as described by CLSI (Clinical and Laboratory Standards Institute, 2017 ), using cells grown 16–18 hr at 37°C on Mueller‐Hinton agar plates, and using the reference strain E. coli ATCC 25922 as a quality control (Clinical and Laboratory Standards Institute, 2017 ). All antibiotic disks (meropenem 10 μg, imipenem 10 μg, cefotaxime 30 μg, ciprofloxacin 5 μg, gentamicin 10 μg, and tetracycline 30 μg) were purchased from Becton Dickinson (Franklin Lakes, NJ). We used the CLSI zone diameter breakpoint values (Clinical and Laboratory Standards Institute, 2017 ) to determine whether our isolates were resistant, intermediate, or sensitive to the different antibiotics tested. For taxa in which the CLSI zone diameter breakpoint values were not available, we used the Enterobacteriaceae values.
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