The Study design was described in our previous publication [9 (link)]. A subset of twelve fecal samples, collected between 2013 and 2015 from 9 patients with AL, were screened for the presence of carbapenemase producing (CP) strains by selecting them on MacConkey agar supplemented with ertapenem (0.5 mg/L) [10 (link)]. Cultivated bacteria were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Microflex, Bruker Daltonics, Bremen, Germany). The resistance phenotype of the isolates was evaluated by testing their susceptibly against sixteen antibiotics on Mueller Hinton agar using disk diffusion methodology according to the European Committee on Antimicrobial Susceptibility testing (EUCAST) guidelines (http://www.eucast.org ). The minimum inhibitory concentration (MIC) of imipenem was determined using Etest method (AB Biodisk, Sweden), the results were interpreted according to the EUCAST breakpoint. The modified Carba NP test method was used to determine a possible carbapenemase production [11 (link)].
E test method
Manufactured by bioMérieux
Sourced in France, Sweden, United Kingdom, United States, Spain
The E-test method is a laboratory test used to determine the antimicrobial susceptibility of microorganisms. It provides a quantitative measurement of the minimum inhibitory concentration (MIC) of a specific antimicrobial agent against a given bacterial strain. The test is performed on agar plates and involves the use of a plastic strip impregnated with a predefined concentration gradient of the antimicrobial agent.
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Lab products found in correlation
Vitek 2 system, by bioMérieux (4 mentions)
Vitek 2 automated system, by bioMérieux (2 mentions)
Meropenem, by bioMérieux (2 mentions)
Imipenem, by bioMérieux (2 mentions)
Vancomycin, by Thermo Fisher Scientific (2 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (2 mentions)
Vitek 2 compact 15, by bioMérieux (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Euromedex (2 mentions)
Isovitalex, by BD (2 mentions)
Vitek ms, by bioMérieux (2 mentions)
Microflex, by Bruker (1 mentions)
Imipenem, by Mast Group (1 mentions)
Vitek 2 compact, by bioMérieux (1 mentions)
Vitek 2 compact system, by bioMérieux (1 mentions)
Brain heart infusion agar medium, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by BD (1 mentions)
Minocycline, by BD (1 mentions)
Chloramphenicol, by BD (1 mentions)
Aztreonam, by BD (1 mentions)
Gentamicin, by BD (1 mentions)
101 protocols using e test method
1
Screening Carbapenemase-Producing Bacteria from Fecal Samples
2
Carbapenem-resistant Enterobacteriaceae Detection
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The active surveillance involved performing a rectal culture using a dry rayon swab. Alternatively, instead of a rectal culture, a culture could be obtained from the perirectal area (if visible stool was present) or from a fecal incontinence bag. All swabs were transported to the central laboratory in liquid Stuart medium (Copan, Italia) within 2 hrs.
China Blue Agar Medium (XFL Medical Sales Co., Ltd, Beijing, People's
Republic of China) was used for the isolation of Enterobacteriaceae from rectal culture samples. Identification and antimicrobial susceptibility testing were performed in the clinical microbiology laboratory using a Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France). Carbapenem (meropenem and/or imipenem) resistance was confirmed by the E-test method according to the manufacturer’s instructions (AB Biodisk, Solna, Sweden). CRE isolates were tested for carbapenemase genes (blaKPC, blaNDM and blaIMP) using PCR.20 (link),21 (link) Multilocus sequence typing (MLST) was performed according to the protocol described on the K. pneumoniae MLST website (www.pasteur.fr/mlst ). Internal fragments of seven housekeeping genes for K. pneumoniae (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified, sequenced, and analyzed. Alleles and sequence types (STs) were determined according to the MLST database (www.pasteur.fr/mlst/Kpneumoniae.html ).
China Blue Agar Medium (XFL Medical Sales Co., Ltd, Beijing, People's
Republic of China) was used for the isolation of Enterobacteriaceae from rectal culture samples. Identification and antimicrobial susceptibility testing were performed in the clinical microbiology laboratory using a Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France). Carbapenem (meropenem and/or imipenem) resistance was confirmed by the E-test method according to the manufacturer’s instructions (AB Biodisk, Solna, Sweden). CRE isolates were tested for carbapenemase genes (blaKPC, blaNDM and blaIMP) using PCR.20 (link),21 (link) Multilocus sequence typing (MLST) was performed according to the protocol described on the K. pneumoniae MLST website (
3
Antimicrobial Susceptibility of MBL-Producing Pseudomonas
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Minimum inhibitory concentrations (MICs) of the 11 antimicrobial agents routinely prescribed in burn centers against the 55 isolates of MBL-producing P. aeruginosa were determined by the E-test method (AB BIODISK, Solna, Sweden), as recommended by the National Committee For Clinical Laboratory Standard Institute (CLSI) (19 (link)). A bacterial suspension from growth in a tryptic soy agar (TSA) plate was prepared in 2 mL of Mueller-Hinton broth (MHB), and the turbidity was adjusted so that it was equivalent to that of a 0.5 McFarland standard. The bacterial suspension was streaked onto a 150-mm-diameter plate containing Mueller-Hinton agar (MHA); the plate was later incubated at 35 to 37℃ in ambient air for 16 to 18 hours. The MIC was read on the basis of the interception of the elliptical zone of growth inhibition with the graded E-test strip according to the manufacturer’s instructions. The antibiotics (Mast Co., UK) consisted of imipenem (10 μg), meropenem (10 μg), cefepime (30 μg), ceftazidime (30 μg), piperacillin/tazobactam (110 μg), ciprofloxacin (5 μg), tobramycin (10 μg), amikacin (30 μg), gentamicin (10 μg), ampicillin (10 μg), and aztreonam (30 μg). American typing collection (ATCC 27853) of P. aeruginosa was used as a control strain to determine antibacterial susceptibility.
4
Antibiotic Susceptibility of MβLs Producing P.aeruginosa
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Minimum inhibitory concentrations (MICs) of the 11 antibiotics, routinely prescribed in burn centers, against the 60 isolates of MβLs producing P.aeruginosa, were determined by the E test method (AB Biodisk, Sweden) as recommended by the National Committee for Clinical Laboratory Standard (CLSI) (18 ). These antibiotics included: imipenem (IMI), meropenem (MEM), cefepime (CPM), ceftazidime (CAZ), piperacillin/tazobactam (PTZ), ciprofloxacin (CIP), tobramycin (TN), amikacin (AK), gentamicin (GM), ampicillin (AP) and aztreonam (ATM). American typing collection (ATCC 27853) of P. aeruginosa was used as a control strain in antibacterial susceptibility determination. Following overnight incubation in Muller-Hinton agar, the MICs breakpoints were interpreted according to the manufacturer’s instructions.
5
Microbiological Culture Sensitivity Assay
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Microbiological culture sensitivity tests were performed for patients while they were admitted in the ICU. A 5–10 ml blood sample was withdrawn from each patient and inoculated into 1 BD BACTEC Plus Aerobic/F® vial and into 1 BD BACTEC Lytic Anaerobic/F® vial. Blood cultures were then incubated in a BACTEC 9240 (Becton Dickinson Diagnostic Instrument Systems, Towson, USA) for 5 days. Other samples for the microbiological culture were taken depending on the site of the infection. The MIC for the isolated organisms to imipenem was determined using the E-test method (AB Biodisk, Solna, Sweden).
6
Antibiotic Susceptibility Profiling of A. baumannii
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For the operation and isolation of all 10 strains of A. baumannii in this study we strictly followed the requirements set by the National Clinical Laboratory Procedures. Ten clinical isolates of A. baumannii frozen in the laboratory were taken out from the ultra-low temperature refrigerator at −80 °C, inoculated on blood plates and was placed in a 37 °C incubator overnight. In the next day, the single bacteria was added into distilled water to 0.5 Meek turbidity.
Bacterial species were identified by using VITEK-2 Compact bacterial instrument (BioMerieux, Lyons, France) following the manufacturer’s instruction.11 (link) Three different methods performed antibiotic susceptibility testing: the sensitivity of piperacillin/tazobactam was determined by the K-B method; the sensitivity of tigecycline was determined by the E-test method (AB Biodisk, Solna, Sweden); the sensitivity of other antimicrobial agents was detected using the VITEK-GN13 drug susceptibility card. The criteria of the susceptibility of the GN13 card and piperacillin/tazobactam were adapted from the Clinical and Laboratory Standards Institute (CLSI;http://clsi.org/standards/ ).
Bacterial species were identified by using VITEK-2 Compact bacterial instrument (BioMerieux, Lyons, France) following the manufacturer’s instruction.11 (link) Three different methods performed antibiotic susceptibility testing: the sensitivity of piperacillin/tazobactam was determined by the K-B method; the sensitivity of tigecycline was determined by the E-test method (AB Biodisk, Solna, Sweden); the sensitivity of other antimicrobial agents was detected using the VITEK-GN13 drug susceptibility card. The criteria of the susceptibility of the GN13 card and piperacillin/tazobactam were adapted from the Clinical and Laboratory Standards Institute (CLSI;
7
Antibiotic Resistance Profiling of H. pylori
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One biopsy specimen each from the antrum and corpus were obtained for H. pylori isolation using previously described culture methods.10 (link),11 (link) The biopsy specimens were cultured on plates containing Brucella chocolate agar with 7% sheep blood and incubated for 4–5 days under microaerobic conditions. The minimal inhibitory concentration (MIC) was determined using the agar dilution test. The H. pylori strains were tested for susceptibility to amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline using the E-test method (AB Biodisk, Solna, Sweden). H. pylori strains had MIC values of ≥0.5, ≥1, ≥1, ≥4, and ≥8 mg/L, which were considered to be the resistance breakpoints for amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline, respectively.22 (link)
8
Antimicrobial Susceptibility Testing Protocol
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Microbial identification and antimicrobial susceptibility tests were performed on a VITEK II system (bioMérieux, Brazil) for the following antimicrobials: aminoglycoside (gentamicin, amikacin), carbapenems (imipenem, meropenem), cephalosporin (cefepime), fluoroquinolone (ciprofloxacin) and penicillin plus β-lactamase inhibitors (piperacillin-tazobactam). Quality-control protocols were used according to the standards of the Clinical and Laboratory Standard Institute [37 , 38 ]. The isolates with intermediate susceptibility were considered as resistant.
The minimum inhibitory concentration (MIC) and the confirmation test of resistance to imipenem (≥8 μg/mL) were performed by the E-test® method, according to the manufacturer’s guidelines (AB Biodisk, Sweden) [38 ]. In addition, resistance to ciprofloxacin was confirmed by broth microdilution method according to Capuano [39 ] with modifications, and the interpretations also were made according to CLSI [38 ], considering resistance to ciprofloxacin ≥4 μg/mL.
The minimum inhibitory concentration (MIC) and the confirmation test of resistance to imipenem (≥8 μg/mL) were performed by the E-test® method, according to the manufacturer’s guidelines (AB Biodisk, Sweden) [38 ]. In addition, resistance to ciprofloxacin was confirmed by broth microdilution method according to Capuano [39 ] with modifications, and the interpretations also were made according to CLSI [38 ], considering resistance to ciprofloxacin ≥4 μg/mL.
9
Antimicrobial Susceptibility Testing for Bacterial Isolates
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Bacterial species were identified by using an VITEK-2 bacterial instrument (BioMerieux, Lyons, France) following the manufacturer's instruction. Antimicrobial susceptibility testing was performed by three different methods: the sensitivity of meropenem and cefotaxime was determined by the disk diffusion method; the sensitivity of tigecycline and polymyxin B was determined by the Etest method (AB Biodisk, Solna, Sweden); the sensitivity of other antimicrobial agents was detected using the VITEK-GN13 drug susceptibility card. The criteria of the susceptibility of the GN13 card, meropenem, and cefotaxime were adapted from the Clinical and Laboratory Standards Institute (CLSI; http://clsi.org/standards/ ). The criteria of the susceptibility of tigecycline were adapted from the United States Food and Drug Administration (http://www.fda.org.uk/sitemap.aspx ).
10
Antibiotic Susceptibility of Pneumococcal Isolates
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For 118 isolates, susceptibility to chloramphenicol, cotrimoxazole erythromycin, and vancomycin was determined by the Kirby–Bauer disc diffusion method, and the results were interpreted according to CLSI 2012 guidelines.27 The minimum inhibitory concentration (MIC) of penicillin and cefotaxime were determined using E-test method (AB Biodisk, Solna, Sweden) as recommended by CLSI. Reference strain with known susceptibility (S. pneumoniae ATCC 49619) was included as control. The MDR organism phenotype was defined as having diminished susceptibility to three or more antibiotics. Interpretation of the results was done according to CLSI 2012 recommendation for meningitis and nonmeningitis cases.
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