Antimicrobial resistance profiles were studied by using the Kirby Bauer technique with the following antibiotics: ampicillin (10 μg), amikacin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamycin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), streptomycin (10 μg), sulfaprim (trimethoprim-sulfamethoxazole; 25 μg) and tetracycline (30 μg), as described elsewhere [56 (link)]. Antimicrobial disks were obtained from Oxoid (United Kingdom). Interpretation of results was carried out by referring to the Clinical & Laboratory Standards Institute (CLSI), 2018 range.
Antimicrobial disks
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Antimicrobial disks are laboratory products used to test the susceptibility of microorganisms to various antimicrobial agents. These disks contain predetermined concentrations of antimicrobial compounds and are placed on agar plates inoculated with the test microorganisms. The resulting zones of inhibition are measured to determine the antimicrobial susceptibility of the tested microbes.
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Lab products found in correlation
Mueller hinton agar (mha), by Thermo Fisher Scientific (5 mentions)
Etest strips, by bioMérieux (4 mentions)
Ciprofloxacin, by Thermo Fisher Scientific (2 mentions)
Cefoxitin, by bioMérieux (2 mentions)
Cefoxitin, by Thermo Fisher Scientific (2 mentions)
Amoxicillin, by Thermo Fisher Scientific (2 mentions)
Ampicillin, by Thermo Fisher Scientific (2 mentions)
Antimicrobial disks, by BD (1 mentions)
Mha plates, by BD (1 mentions)
Levofloxacin, by Merck Group (1 mentions)
Nalidixic acid, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Enrofloxacin, by Bayer (1 mentions)
Marbofloxacin, by Vetoquinol (1 mentions)
Lennox broth, by Merck Group (1 mentions)
Iso sensitest broth, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Nalidixic acid, by Thermo Fisher Scientific (1 mentions)
Etest, by bioMérieux (1 mentions)
Bhi broth, by Bio-Rad (1 mentions)
38 protocols using antimicrobial disks
1
Antimicrobial Resistance Profiling Protocol
2
Antimicrobial Susceptibility of Streptococcus Isolates
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We isolated 11 Streptococcus strains from collected fecal samples and tested against 13 antimicrobials (Additional file 3 : Table S2 a,b). The susceptibilities of all the isolates were determined independently against beta-lactam (Penicillin 10 μg, Ampicillin 10 μg, Ceftriaxone 5 μg); Macrolides (Erythromycin 15 μg, Azithromycin 15 μg, Clarithromycin 5 μg); Lincosamide (Clindamycin 2 μg); Quinolones (Levofloxocin 5 μg, Ofloxacin 5 μg); Ansamycin (Rifampin 5 μg); Glycopeptides (Vancomycin 30 μg); Miscellaneous (Chloramphenicol 30 μg); Carbapenems (Meropenem 10 μg) using the standard Kirby–Bauer disk diffusion method [90 (link)]. Colony suspension, equivalent to a 0.5 McFarland standard, were prepared using colonies from an overnight (18-20 h) sheep blood agar plate at 35±2 °C.Antimicrobial disks (Oxoid Ltd., Basingstoke, UK) were placed on the MHA plates (Becton Dickinson, Franklin Lakes, NJ) with 5% sheep blood and incubated aerobically at 35±2 °C for 20–24 h. The inhibition zones’ diameter surrounding the Antimicrobial disks was interpreted according to Clinical and Laboratory Standards Institute guidelines (CLSI, M100, 30th Edition, 2020). Quality control for susceptibility testing was done using S. pneumoniae ATCC® 49619.
3
Antimicrobial Susceptibility Testing of Pseudomonas and E. coli
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Antimicrobial susceptibility analyses were performed by the disk diffusion test, according to the Clinical Laboratory of Standard Institute guidelines [16 ]. Eleven different antimicrobial disks were used (Oxoid Limited, Basingstoke, UK) for testing: piperacillin, piperacillin-tazobactam, cefepime, ceftazidime, ciprofloxacin, gentamicin, amikacin, tobramycin, meropenem, and imipenem.
The minimum inhibitory concentrations (MICs) for fluoroquinolone antimicrobials were determined as indicated by the CLSI broth microdilution method. The quinolones and fluoroquinolones used were nalidixic acid (Sigma-Aldrich, Germany), ciprofloxacin (Sigma-Aldrich), levofloxacin (Sigma-Aldrich), enrofloxacin (Bayer Vital GmbH, Leverkusen, Germany), and marbofloxacin (Vetoquinol, Lure, France). P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control strains. The breakpoints for ciprofloxacin (≥4 μg/mL) and levofloxacin (≥8 μg/mL) were followed based on CLSI guidelines, whereas the breakpoints for nalidixic acid (≥32 μg/mL) was followed as described by Rubin et al., 2008 and for enrofloxacin (≥4 μg/mL) and marbofloxacin (≥4 μg/mL) as described by Pintarić et al., 2017.
The minimum inhibitory concentrations (MICs) for fluoroquinolone antimicrobials were determined as indicated by the CLSI broth microdilution method. The quinolones and fluoroquinolones used were nalidixic acid (Sigma-Aldrich, Germany), ciprofloxacin (Sigma-Aldrich), levofloxacin (Sigma-Aldrich), enrofloxacin (Bayer Vital GmbH, Leverkusen, Germany), and marbofloxacin (Vetoquinol, Lure, France). P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control strains. The breakpoints for ciprofloxacin (≥4 μg/mL) and levofloxacin (≥8 μg/mL) were followed based on CLSI guidelines, whereas the breakpoints for nalidixic acid (≥32 μg/mL) was followed as described by Rubin et al., 2008 and for enrofloxacin (≥4 μg/mL) and marbofloxacin (≥4 μg/mL) as described by Pintarić et al., 2017.
4
Bacterial Growth and Antibiotic Evaluation
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Strains used in this study are listed in Table 7 . Bacterial strains were grown overnight at 37°C in Lennox broth (Sigma-Aldrich, UK) or Iso-Sensitest broth (Oxoid, UK). All chemicals and antibiotics were supplied by Sigma-Aldrich, UK. Antimicrobial disks were supplied by Oxoid, UK.
5
Antimicrobial Resistance Profiling of MRSA
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To determine the antimicrobial resistance of MRSA isolates, the disk diffusion method was performed and interpreted according to the CLSI standard (M100-S27, 2017). The tested antimicrobial disks (Oxoid Ltd., Hampshire, UK) included penicillin (10 Units), erythromycin (15 µg), clindamycin (2 µg), trimethoprim-sulfamethoxazole (25 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), gentamicin (10 µg), rifampin (5 µg), tetracycline (30 µg), cephazolin (30 µg), fosfomycin (50 µg), and linezolid (30 µg).
6
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility testing was performed for all isolates using the disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute – CLSI.18 The following antimicrobial disks (Oxoid) were used: nalidixic acid (30 μg), amoxicillin–clavulanic acid (20/10 μg), amikacin (30 μg), ampicillin (10 μg), aztreonam (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), cefepime (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), streptomycin (10 μg), gentamicin (10 μg), imipenem (10 μg), trimethoprim–sulfamethoxazole (1.25/23.75 μg), sulfonamide (250 μg), and tetracycline (30 μg). Categorization of the diameter of halos in susceptible, intermediate or resistant followed CLSI recommendations.18
Minimum inhibitory concentrations (MIC) were determined for nalidixic acid and ciprofloxacin by Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's recommendations. The range of MIC of ciprofloxacin for Salmonella was recently changed to susceptible: ≤0.06 μg/mL; intermediate susceptible: 0.12–0.5 μg/mL; resistant: ≥1 μg/mL.18
E. coli ATCC 25922 and E. coli ATCC 35218 were used as reference strains for antimicrobial susceptibility testing.
Minimum inhibitory concentrations (MIC) were determined for nalidixic acid and ciprofloxacin by Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's recommendations. The range of MIC of ciprofloxacin for Salmonella was recently changed to susceptible: ≤0.06 μg/mL; intermediate susceptible: 0.12–0.5 μg/mL; resistant: ≥1 μg/mL.18
E. coli ATCC 25922 and E. coli ATCC 35218 were used as reference strains for antimicrobial susceptibility testing.
7
Antimicrobial Susceptibility Testing of Bacterial Isolates
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Antimicrobial susceptibility tests were performed on the Mueller-Hinton agar (Oxoid, Ltd) using the Kirby-Bauer disk diffusion method as described by the Clinical and Laboratory Standards Institute (CLSI).31 Three to five pure colonies were picked with a sterile loop and mixed with sterile normal saline (0.85% NaCl) to prepare a uniform suspension equivalent to 0.5 McFarland standards. The suspension was uniformly spread onto a Mueller-Hinton agar plate using a sterile cotton swab. The plate was left at room temperature for 3 to 5 minutes to dry. The antimicrobial disks (Oxoid, Ltd) tested were ampicillin (10 μg), amoxicillin (10 μg), chloramphenicol (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), nalidixic acid (30 μg), sulfamethoxazole-trimethoprim (23.75/1.25 µg), and tetracycline (30 μg). After incubating the plates at 37°C for 24 hours, the zone of inhibition including the disks was measured using a digital caliper to the nearest whole millimeters and interpreted as sensitive, intermediate, or resistant based on CLSI31 interpretive breakpoints. Multidrug resistance (MDR) was defined as simultaneous resistance of the isolates to 2 or more classes of antimicrobial agents.32 (link)
8
Antimicrobial Susceptibility Testing of Enterococcus
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Enterococcus spp. isolates were tested for antimicrobial susceptibility against a panel of 12 antimicrobials by the disk diffusion method (Kirby Bauer Test) as described by the Clinical and Laboratory Standards Institute (CLSI 2018 , 2021). The following antimicrobials were tested: amikacin 30 mg, amoxicillin/clavulanic acid 30 (20 + 10) mg, ampicillin 10 μg, ceftriaxone 30 μg, chloramphenicol 30 μg, ciprofloxacin 5 μg, kanamycin 30 μg, sulphamethoxazole 25 μg, sulphamethoxazole/ trimethoprim 25 μg, tetracycline 30 μg, ticarcillin 75 μg, vancomycin 30 μg. This antimicrobial panel was selected to test the major groups of antimicrobials. Briefly, frozen isolates were thawed and cultured in BHI broth (Bio-Rad) at 35 to 37°C for 24 h. A portion of the culture broth was inoculated into 6 mL of 0.9% sterile physiological saline solution until a turbidity of 0.5 McFarland was reached (1.0 for vancomycin (Wongthong et al. 2015 (link))). Using a sterile swab, the solution was spread on Muller-Hinton agar plates (Oxoid). Antimicrobial disks (Oxoid) were placed on Muller-Hinton agar plates which were incubated at 37°C for 18 to 24 h. At the end of incubation, the diameters of the growth inhibitory zones were measured, and these were interpreted using specific CLSI tables whereby the bacterium is classified as susceptible, intermediately susceptible or resistant (CLSI 2021).
9
Antimicrobial Resistance Profiling of Isolates
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Disk diffusion method was employed to test the resistance of selected isolates to 21 antimicrobials, following CLSI guidelines [24 ]. The tested antimicrobial disks (Oxoid Ltd., Hampshire, United Kingdom) were: amoxicillin–clavulanic acid (30 μg), ampicillin (10 μg), cefalotin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), enrofloxacin (5 μg), erythromycin (10 μg), gentamicin (10 μg), linezolid (30 μg), neomycin (10 μg), nitrofurantoin (300 μg), oxacillin (1 μg), quinupristin–dalfopristin (15 μg), rifampicin (30 μg), streptomycin (10 μg), teicoplanin (30 μg), tetracycline (30 μg), tigecycline (15 μg), trimethoprim (5 μg),and vancomycin (30 μg). Results were interpreted following CLSI and EUCAST breakpoint tables [25 ,26 ]. Isolates resistant to streptomycin or gentamicin in disk diffusion test were examined for high level aminoglycoside resistance (HLAR) following CLSI guidelines [26 ] and employing high concentration of gentamicin (500 μg/mL) and streptomycin (1000 μg/mL). Minimum Inhibitory Concentration (MIC) for vancomycin and ampicillin was evaluated in all disk diffusion test resistant and intermediate isolates [26 ,27 ].
On the basis of phenotypic resistance patterns, isolates were classified as multidrug-resistant (MDR), extensively drug-resistant (XDR) or pandrug-resistant (PDR) [28 (link)].
On the basis of phenotypic resistance patterns, isolates were classified as multidrug-resistant (MDR), extensively drug-resistant (XDR) or pandrug-resistant (PDR) [28 (link)].
10
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method as described by the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute [CLSI], 2016 ). Antimicrobial disks (Oxoid Ltd., Basingstoke, United Kingdom) used for testing contained 10 μg amoxicillin, 10 μg ampicillin, 30 μg cefotaxime, 30 μg cefuroxime sodium, 30 μg cephazolin, 30 μg chloramphenicol, 5 μg ciprofloxacin, 30 μg nalidixic acid, 10 μg norfloxacin, 10 μg streptomycin, 10 μg gentamicin, 300 μg nitrofurantoin, 300 μg sulfonamide and 30 μg tetracycline. Escherichia coli strain ATCC 25922 was used as a reference strain. The standard break points were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute [CLSI], 2016 ).
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