A modification of the standard disk diffusion method (rAST) was evaluated for the detection of antimicrobial resistance. The rAST followed CLSI standards in all aspects, except for the inoculum preparation. CLSI guideline recommends preparing the inoculum for AST with a direct suspension of isolated colonies selected from 18–24 h agar plate incubation [6 ]. In this study, we used colonies selected from a rapid culture grown (4–6 h agar plate incubation). The inoculum was prepared from the rapid culture grown on a 150 mm Mueller–Hinton Agar (bioMérieux, France), and then, disks were applied and plates incubated at 35°C ± 1° and incubated for 18 h. The following antimicrobial agents were evaluated for GN: amikacin 30 μg, amoxicillin-clavulanate 20/10 μg, ampicillin 10 μg, ampicillin-sulbactam 10/10 μg, cefepime 30 μg, ceftazidime 30 μg, cefuroxime 30 μg, ciprofloxacin 5 μg, gentamicin 10 μg, meropenem 10 μg, piperacillin-tazobactam 100/10 μg, and trimethoprim-sulfamethoxazole 23.75/1.25 μg (Oxoid®, Thermo-Fisher, USA). For GP, cefoxitin 30 μg, clarithromycin 15 μg, clindamycin 2 μg, doxycycline 30 μg, erythromycin 15 μg, gentamicin 10 μg, levofloxacin 5 μg, rifampicin 5 μg, and trimethoprim-sulfamethoxazole 23.75/1.25 μg (Oxoid®) were evaluated. The inhibition zones were analyzed after 18–24 h, and the results were interpreted by CLSI proposed breakpoints [6 ].
Trimethoprim sulfamethoxazole
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Trimethoprim-sulfamethoxazole is a combination antibiotic medication used in the treatment of various bacterial infections. It consists of two active ingredients, trimethoprim and sulfamethoxazole, which work together to inhibit the growth and reproduction of bacteria.
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Lab products found in correlation
Ciprofloxacin, by Thermo Fisher Scientific (84 mentions)
Tetracycline, by Thermo Fisher Scientific (79 mentions)
Gentamicin, by Thermo Fisher Scientific (76 mentions)
Chloramphenicol, by Thermo Fisher Scientific (54 mentions)
Ampicillin, by Thermo Fisher Scientific (54 mentions)
Erythromycin, by Thermo Fisher Scientific (47 mentions)
Cefoxitin, by Thermo Fisher Scientific (44 mentions)
Ceftriaxone, by Thermo Fisher Scientific (43 mentions)
Imipenem, by Thermo Fisher Scientific (39 mentions)
Clindamycin, by Thermo Fisher Scientific (38 mentions)
Ceftazidime, by Thermo Fisher Scientific (36 mentions)
Cefotaxime, by Thermo Fisher Scientific (36 mentions)
Meropenem, by Thermo Fisher Scientific (34 mentions)
Amikacin, by Thermo Fisher Scientific (34 mentions)
Nalidixic acid, by Thermo Fisher Scientific (29 mentions)
Cefepime, by Thermo Fisher Scientific (27 mentions)
Nitrofurantoin, by Thermo Fisher Scientific (24 mentions)
Norfloxacin, by Thermo Fisher Scientific (23 mentions)
Amoxicillin clavulanic acid, by Thermo Fisher Scientific (22 mentions)
Aztreonam, by Thermo Fisher Scientific (22 mentions)
123 protocols using trimethoprim sulfamethoxazole
1
Rapid Antimicrobial Susceptibility Testing Protocol
2
Antibiotic Susceptibility Profiling of Isolates
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All the identified isolates were subjected to susceptibility testing against amoxicillin-clavulanic acid (30 µg), ampicillin (10 µg), cefepime (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cefuroxime (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), imipenem (10 µg), meropenem (10 µg), nitrofurantoin (10 µg), piperacillin (100 µg), sulfamethoxazole-trimethoprim (1.25/23.75 µg), and tetracycline (30 µg) (Oxoid, UK) using the Kirby-Bauer method on Mueller Hinton Agar (MHA) (Oxoid, UK). Zone sizes from the Clinical and Laboratory Standard Institute guidelines were employed to interpret the results. Bacterial isolates which were resistant to three or more antibiotics from different classes were considered as MDR.24
3
Antimicrobial Resistance Profiling of Staphylococcal Isolates
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All staphylococcal isolates were tested against 13 different antimicrobials of various groups using disk diffusion method [19 ]. The antimicrobials used were ampicillin (10 μg), ciprofloxacin (5 μg), gentamicin (30 μg), tetracycline (30 μg), erythromycin (15 μg), oxacillin (5 μg), amoxicillin (10 μg), cefaclor (30 μg), sulfamethoxazole- trimethoprim (23.75+1.25 μg), cefoxitin (10 μg), ceftriaxone (10 μg), penicillin (10 IU), and streptomycin (100 μg) (Oxoid, Basingstoke, UK). For each isolate, the zone of inhibition was measured and interpreted as susceptible (S), intermediate (I), and resistant (R) according to CLSI guidelines [19 ]. Isolates resistant to ≥3 classes of antimicrobials were considered as MDR [5 ].
4
Antimicrobial Susceptibility Testing Protocol
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The antimicrobial susceptibility testing of the isolated bacterial species was determined by Kirby-Bauer disk diffusion method22 except against vancomycin and polymyxin B where antimicrobial susceptibility was determined by the broth microdilution method.23 The selection of the antimicrobial agents was based on the type of the organism being tested, and the results were interpreted following the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial susceptibility Testing (EUCAST) interpretive criteria.24 ,25 The antibiotic disks included in the study were gentamicin 10 µg, piperacillin/tazobactam 100 µg/10 µg, imipenem 10 µg, meropenem 10 µg, cefazolin 30 µg, ceftazidime 30 µg, cefepime 30 µg, cefoxitin 30 µg, ciprofloxacin 5 µg, sulfamethoxazole/trimethoprim 23.75 µg/1.25 µg, tigecycline 15 µg, aztreonam 30 µg, chloramphenicol 30 µg, doxycycline 30 µg, fusidic acid 10 µg, clindamycin 2 µg, erythromycin 15 µg, linezolid 30 µg and quinupristin/dalfopristin 15 µg (All from Oxoid Ltd., Hampshire, UK).
5
Antimicrobial Susceptibility Testing of S. pneumoniae
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In vitro susceptibility tests were performed using disk diffusion and the following antibiotics were used; erythromycin, clindamycin, levofloxacin, vancomycin, tetracycline, sulfamethoxazole-trimethoprim and chloramphenicol (Oxoid, UK). The minimum inhibitory concentrations for penicillin and ceftriaxone were determined by E-test (AB Biodisk, Solna, Sweden). All tests were performed following the United States Clinical and Laboratory Standards Institute (CLSI) recommendations, and CLSI M100-S25 version of the antibiotic susceptibility breakpoints for S. pneumoniae was adopted as criteria for determining drug resistance [20 ]. S. pneumoniae ATCC 49619 was used as the quality-control strain. Isolates not susceptible to three or more classes of antimicrobials were considered multidrug-resistant (MDR).
6
Antibiotic Resistance Profiling of A. baumannii
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Antibiotic susceptibility testing (AST) was performed by the Kirby–Bauer disc diffusion method using a 0.5 McFarland bacterial suspension spread over Mueller Hinton agar (Oxoid Ltd. Bashingstore Hampshire, UK). Seventeen antibiotic discs were utilized to determine the multidrug-resistant A. baumannii according to Clinical and Laboratory Standards Institute guidelines [17 ] (CLSI-2015). The susceptibility of the isolated strains was tested against amikacin (30 µg), aztreonam (30 µg), Cefepime (30 µg), Cefoperazone (75 µg), ceftazidime (30 µg), levofloxacin (5 µg) Ceftriaxone (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), imipenem (10 µg), (5 µg), meropenem (10µg), piperacillin-tazobactam (100/10 µg), piperacillin (100 µg), tetracycline (30 µg), tobramycin (10 µg), sulfamethoxazole-trimethoprim (2.75/1.25 µg), and colistin (10 µg) (all from Oxoid, UK). The strains were recorded as sensitive, intermediate, or resistant based upon CLSI-2017 guidelines for disk diffusion method–Mueller Hinton agar; Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control (QC). The QC strains for antimicrobial testing were as is recommended in the CLSI-2015 guidelines [17 ].
7
Antibiotic Susceptibility Testing by Disk Diffusion
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Antimicrobial susceptibility test by the disk diffusion method was performed to determine the resistance patterns of the isolates to 17 antibiotics: cefotaxime, ceftriaxone, ceftazidime, aztreonam, ceftazidime/clavulanate, cefotaxime/clavulanate, cephalothin, cefuroxime sodium, cefepime, gentamicin, amikacin, ciprofloxacin, tetracycline, ampicillin, amoxicillin/clavulanate, piperacillin, piperacillin/tazobactam, imipenem, meropenem, ertapenem, and sulfamethoxazole/trimethoprim [Oxoid, England; disc contents according to Clinical and Laboratory Standards Institute (CLSI) guidelines]. All antimicrobial testing was performed on Mueller-Hinton agar by the flooding technique and data interpreted according to the CLSI guidelines.
8
Serotyping and Antimicrobial Susceptibility of Vibrio cholerae
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The isolates were serotyped with polyvalent O1 antiserum and monovalent Inaba and Ogawa antisera (Denka Seiken, Tokyo, Japan). Biotypes were determined by (i) susceptibility to polymyxin B, (ii) haemolysis of sheep erythrocytes and (iii) the Voges-Proskauer test, which measured the production of acetylmethylcarbinol.
Antimicrobial susceptibility testing was performed with the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. The Escherichia coli reference strain ATCC 25922 served as a control. Isolates were tested against 7 antimicrobial drugs as follows: ampicillin (10 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), gentamycin (30 μg), nalidixic acid (30 μg), sulfamethoxazole/trimethoprim (1.25 μg + 23.75 μg) and tetracycline (30 μg)(all Oxoid, Basingstoke, United Kingdom). Diameters were interpreted according to the 2015 Clinical and Laboratory Standards Institute (CLSI) guidelines as resistant, susceptible or intermediate (http://www.clsi.org ). Intermediate isolates will be referred to as reduced susceptible in the following. When no interpretive criteria for V. cholerae were available, breakpoints for Enterobacteriaceae according to the 2015 European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines were applied (http://www.eucast.org ).
Antimicrobial susceptibility testing was performed with the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. The Escherichia coli reference strain ATCC 25922 served as a control. Isolates were tested against 7 antimicrobial drugs as follows: ampicillin (10 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), gentamycin (30 μg), nalidixic acid (30 μg), sulfamethoxazole/trimethoprim (1.25 μg + 23.75 μg) and tetracycline (30 μg)(all Oxoid, Basingstoke, United Kingdom). Diameters were interpreted according to the 2015 Clinical and Laboratory Standards Institute (CLSI) guidelines as resistant, susceptible or intermediate (
9
Antimicrobial Susceptibility Testing of Enterobacterales and S. aureus
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For all Enterobacterales and S. aureus, antimicrobial susceptibility testing was performed using Kirby-Bauer disk diffusion method on Mueller-Hinton agar according to the 2019 European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (EUCAST: EUCAST, 2019 ). Enterobacterales were tested against ampicillin, cefotaxime, ceftazidime, meropenem, ciprofloxacin, gentamicin, tigecycline and sulfamethoxazole/trimethoprim (all Oxoid, Basingstoke, United Kingdom). ciprofloxacin resistance was defined as a MIC > 0.06 mg/L for S. enterica, confirmed by E-test (Oxoid, Basingstoke, United Kingdom) and a MIC > 1 mg/L for other Enterobacterales. A positive ESBL phenotype was confirmed by the double-disk diffusion test with cefotaxime and ceftazidime alone and in combination with clavulanic acid (Becton, Dickinson and Company, Sparks, MD, United States) as described before by the EUCAST (EUCAST, 2020 : Resistance mechanisms). S. aureus isolates were tested against penicillin, cefoxitin, clindamycin, erythromycin, ciprofloxacin, tetracycline, sulfamethoxazole/trimethoprim, tigecycline, gentamicin, rifampicin, linezolid, teicoplanin and vancomycin (all Oxoid, Basingstoke, United Kingdom).
10
Antibiotic Susceptibility of K. pneumoniae
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The susceptibility of K. pneumoniae isolates to different antibiotics was tested using the modified Kirby Bauer’s disc diffusion method on Mueller–Hinton agar plates (Oxoid Ltd., Basingstoke, UK). Antibiotic discs including sulfamethoxazole/trimethoprim (SXT; 25 μg), ampicillin/sulbactam (SAM; 20 μg), piperacillin/tazobactam (TZP; 110 μg), cefepime (FEP; 30 μg), ceftriaxone (CRO; 30 μg), ceftazidime (CAZ; 30 μg), cefotaxime (CTX; 30 μg), aztreonam (ATM; 30 μg), ciprofloxacin (CIP; 10 μg), levofloxacin (LEV; 10 μg), gentamicin (CN; 10 μg), amikacin (AK; 30 μg), imipenem (IPM; 10 μg), and meropenem (MEM; 10 μg) were purchased from Oxoid Ltd. (Basingstoke, UK), while CZA discs (50 μg) were obtained from Liofilchem®, Italy. The CRKP isolates were preliminary identified if they showed resistance (zone diameter ≤19 mm) to at least one of the used carbapenems (imipenem and meropenem). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 (Naval Medical Research Unit Three, Cairo, Egypt; NAMRU-3) were included as quality control strains. All susceptibility results were interpreted as per the recommendations of the Clinical and Laboratory Standards Institute (CLSI).13
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