Susceptibility testing for relevant anti-pseudomonal antibiotics was carried out using the disk diffusion method (Oxoid, Basingstoke, UK) and E-tests (Liofilchem, Roseto degli Abruzzi, Italy) on Mueller-Hinton agar plates (bioMérieux, Marcy-l’Étoile, France) including amikacin, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, levofloxacin and meropenem. Colistin susceptibility was performed using the broth microdilution assay in cation-adjusted Mueller-Hinton broth (Merlin Diagnostika GmbH, Bremen, Germany). Interpretation of the results was based on the standards and breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) v. 11.0 [38 ]. Results indicating “susceptible, increased exposure (I)” were grouped with and reported as susceptible (S) [39 ]. Classification of the isolates as MDR (resistance to at least one agent in ≥3 antibiotic groups) was based on Magiorakos et al. [40 (link)]. A multiple antibiotic resistance (MAR) index—ranging between 0 and 1—was calculated by dividing the total number of detected resistance to antimicrobials for each isolate by the total number of tested antimicrobials [41 (link)].
Disk diffusion method
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The disk diffusion method is a laboratory technique used to determine the susceptibility of bacteria to antimicrobial agents. It involves placing a bacterial inoculum on a culture medium and then placing antimicrobial-impregnated disks on the surface. The size of the resulting zone of inhibition around each disk is measured and used to categorize the bacteria as susceptible, intermediate, or resistant to the tested antimicrobials.
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Lab products found in correlation
Mueller hinton agar plate, by bioMérieux (1 mentions)
E test, by Liofilchem (1 mentions)
Etest, by bioMérieux (1 mentions)
Vitek 32 automated microbial analyzer, by bioMérieux (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
E test method, by bioMérieux (1 mentions)
E test strip, by bioMérieux (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
10 protocols using disk diffusion method
1
Antibiotic Susceptibility Profiling of Pseudomonas
2
Pneumococcal Carriage Surveillance Protocol
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Nasopharyngeal swabs were obtained using rayon-tipped swabs and transported in Amies-charcoal media (COPAN, Italy). Swabs were inoculated on 5% sheep blood agar with gentamicin (5 mg/liter) and incubated at 35°C in 5% CO2 for 48 hours. Identification of S. pneumoniae was performed using the optochin test (Oxoid, Basingstoke, UK) and, in case of doubt, a DNA hybridization test (Accuprobe, Gen-Probe Inc., San Diego, CA, USA). Antimicrobial susceptibility tests were performed using disk diffusion method (Oxoid, UK) and E-test (bioMérieux, France) and interpreted according to EUCAST 2012. Serotyping of S.pneumoniawas done with a multiplex-PCR which covers 36 serotypes [5] , [6] (link). Control strains were included in all analyses.
Data on demography, house sanitation (crowding, smoke exposure from cigarette and mosquito coils), and water and food hygiene, were recorded using a questionnaire that was developed to identify determinants of carriage. Crowding was defined to be present when the ratio of total bedroom space to the number of family members was less than 4 m2[7] . Water hygiene was defined as poor when water other than tap or bottled water was used by the family. Food hygiene was considered poor if the family consumed street food.
Data on demography, house sanitation (crowding, smoke exposure from cigarette and mosquito coils), and water and food hygiene, were recorded using a questionnaire that was developed to identify determinants of carriage. Crowding was defined to be present when the ratio of total bedroom space to the number of family members was less than 4 m2[7] . Water hygiene was defined as poor when water other than tap or bottled water was used by the family. Food hygiene was considered poor if the family consumed street food.
3
Antimicrobial Resistance Profiling of SDSD Strains
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All SDSD strains were tested for antimicrobial resistance by disk diffusion method (Oxoid Ltd., Basingstoke, United Kingdom), and the determination of minimum inhibitory concentration (MIC) follows the guidelines from the Clinical and Laboratory Standards Institute (CLSI) for antimicrobial susceptibility tests for bacteria isolated from animals (CLSI M31-A3, 2008). The following antimicrobials were selected for testing, based on several criteria: (a) licensing for mastitis treatment in cattle—amoxicillin–clavulanic acid 30 μg (AMC), gentamicin 10 μg (CN), penicillin 10 units (P), pirlimycin 2 μg (PRL), and streptomycin 10 μg (S); and (b) use in human medicine—chloramphenicol 30 μg (CHL), erythromycin 15 μg (ERY), tetracycline 30 μg (TET), and vancomycin 30 μg (VA). The results were interpreted according to the CLSI guidelines (CLSI M31-A3, 2008). The antimicrobial manufacturers’ instructions were followed when CLSI guidelines were not available. The reference strain Staphylococcus aureus ATCC 29213 was used for quality control for both methods.
4
Pathogen Isolation, Identification, and Antibiotic Susceptibility
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Pathogen isolation and culture were carried out based on the National Clinical Testing Protocols (China), and suspected strains were identified using a VITEK 32 automated microbial analyzer (BioMerieux, Craponne, France). Drug susceptibility was detected using the minimum inhibitory concentration (MIC) and disk diffusion method (Oxoid Ltd, UK) and were interpreted in reference to the Clinical and Laboratory Standards Institute (CLSI, 2018).
5
Antimicrobial Susceptibility Testing for Bacterial Isolates
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AST was performed by using the AST-238 card, whose results were evaluated with the VITEK® 2 (BioMérieux, Marcy-I'Etoile, France) system. The following antibiotics were tested: amikacin (AST-N054 only), ampicillin, amoxicillin/clavulanic acid, aztreonam (AST-N054 only), cefalexin, cefepime, cefotaxime, ceftazidime, cefoxitin, cefuroxime, ciprofloxacin, norfloxacin, ertapenem, gentamicin, meropenem, nalidixic acid, nitrofurantoin, piperacillin, piperacillin/tazobactam, and trimethoprim. Clinical Laboratory Standards Institute (CLSI) breakpoints were used for the interpretation of susceptibility testing results. Isolates were classified as susceptible (S), intermediately resistant (I) or resistant (R) to the aforementioned antimicrobials, respectively, according to the following MIC breakpoints (µg/mL): ampicillin, ≤ 8, 16, ≥ 32; amoxicillin-clavulanate, ≤ 8/4, 16/8, ≥ 32/16; cefuroxime axetil, ≤ 4, 8-16, ≥ 32; norfloxacin, ≤ 4, 8, ≥ 16; ciprofloxacin, ≤ 1, 2, ≥ 4; cotrimoxazole, ≤ 2/38, ≥ 4/76; nitrofurantoin, ≤ 32, 64, ≥ 128; fosfomycin was evaluated by disk diffusion method (Oxoid, Cambridge, UK). The isolates were screened for ESBL production through chromID® ESBL agar plate test (BioMérieux, Marcy l’Étoile, France).
6
Antibiotic Resistance Profiling of Bacterial Isolates
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A. hydrophila, S. aureus, and MRSA isolates were evaluated for antibiotic resistance using the disk diffusion method (Oxoid, UK) in accordance with the recommendations of Clinical and Laboratory Standard Institute guidelines (CLSI, 2013 ). The 10 antibiotics tested (bioMérieux F6980, Marcy-l’Étoile, France) were ampicillin (AM, 10 µg), chloramphenicol (C, 30 µg), ciprofloxacin (CIP, 5 µg), enrofloxacin (ENR, 5 µg), erythromycin (ERY, 15 µg), nalidixic acid (NA, 30 µg), gentamicin (GN, 10 µg), amoxicillin (AXE, 25 µg), tetracycline (TE, 30 µg), and trimethoprim/sulfamethoxazole (SXT, 25 µg). Bacterial isolates were classified as resistant (R), intermediate (I), or susceptible (S) by measurement of inhibition zone diameters as prior criteria of CLSI guidelines. Multidrug-resistant isolates demonstrated resistance to at least one antimicrobial drug in three or more different antimicrobial classes (Magiorakos et al., 2012 (link)). Furthermore, the multiple antibiotic resistance (MAR) index for isolates was determined using the formula a/b, where “a” represents the number of antibiotics to which an isolate was resistant and “b” represents the total number of drugs tested (Krumperman, 1983 (link)).
7
Antibiotic Susceptibility Screening of S. aureus
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S. aureus isolates were screened for their susceptibility to six antibiotics (erythromycin, 15 μg; ampicillin, 10 μg; cefoxitin, 30 μg; amoxicillin/clavulanic acid, 20/10 μg; linezolid, 30 μg; and clindamycin, 2 μg) using the disk diffusion method (Oxoid Ltd, Basingstoke, UK) following the Clinical Laboratory Standards Institute (CLSI) guidelines [11 ]. Disk diffusion analysis of cefoxitin resistance to detect MRSA was performed according to CLSI recommendations.
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S. aureus isolates were screened for their susceptibility to six antibiotics (erythromycin, 15 μg; ampicillin, 10 μg; cefoxitin, 30 μg; amoxicillin/clavulanic acid, 20/10 μg; linezolid, 30 μg; and clindamycin, 2 μg) using the disk diffusion method (Oxoid Ltd, Basingstoke, UK) following the Clinical Laboratory Standards Institute (CLSI) guidelines [11 ]. Disk diffusion analysis of cefoxitin resistance to detect MRSA was performed according to CLSI recommendations.
8
Antimicrobial Susceptibility of Pneumococcal Isolates
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The antimicrobial susceptibility of pneumococcal isolates was assessed by the disk diffusion method (Oxoid, USA) for erythromycin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. Meanwhile, the minimal inhibitory concentrations (MIC) of penicillin, ceftriaxone, and cefotaxime were determined using the E-test method (BioMérieux, France). Both methods followed the CLSI procedures and interpretation guidelines, taking into consideration the different MIC criteria for isolates from meningitis and non-meningitis39 . Both assays were tested on Mueller Hinton Agar (Isolab) with 5% sheep blood, incubated at 37 °C in 5% CO2. Isolates resistant to three or more antimicrobial agents were defined as multidrug-resistant (MDR)40 (link).
9
Antibiotic Susceptibility Profiling of GBS
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The minimum inhibitory concentrations (MICs) of penicillin, ceftriaxone, erythromycin, clindamycin, clarithromycin, levofloxacin, and azithromycin were determined using Etest strips (AB Biodisk, Sweden). Susceptibilities to telithromycin and tetracycline were determined by disk diffusion method (Oxoid). The breakpoints adopted were in accordance with the 2012 criteria set by the Clinical and Laboratory Standards Institute (CLSI). The breakpoint for telithromycin was determined by comparison with the quality control culture Streptococcus pneumoniae ATCC 49619. GBS isolates were considered multidrug-resistant if they were not susceptible to three or more classes of antimicrobials tested in this study.
All antibiotic susceptibility data were analyzed using WHONET 5.6 software, as recommended by the World Health Organization (WHO).
All antibiotic susceptibility data were analyzed using WHONET 5.6 software, as recommended by the World Health Organization (WHO).
10
Antimicrobial Resistance Profiling of Listeria monocytogenes
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L. monocytogenes strains were subjected to phenotypical analysis to characterize their resistances against 12 antimicrobials (10 mg ampicillin, 10 units penicillin G, 1 mg oxacillin, 2 mg clindamycin, 15 mg erythromycin, 10mg gentamicin, 10 mg imipenem, 5 mg rifampin, 30 mg chloramphenicol, 30 mg tetracycline, 25 mg trimethoprim/sulfamethoxazole, and 30 mg vancomycin) using the disk diffusion method (Oxoid). Cultures were transferred to brain and heart infusion (Oxoid), incubated at 35°C overnight, and diluted in 0.85% NaCl (w/v) until the turbidity was similar to 0.5 MacFarland. Diluted cultures were swabbed onto the surface of the Mueller-Hinton agar (Oxoid), and the antimicrobial disks were added (three disks per plate). After incubation at 35°C for 18 and 24 hr, the results for each antimicrobial agent were recorded and their resistance profiles were classified as sensitive, intermediate, and resistant, as described by Cockerill 10 to Staphylococcus spp. Reference strain Staphylococcus aureus ATCC 25923 was used as the control.
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