Vitreous samples from patients were investigated using an institutional protocol as described earlier.18 (link) Briefly, the sample was inoculated onto an array of bacterial and fungal media, which were incubated at 37°C and 25°C, respectively, for 7 days. Following growth on culture media, P. aeruginosa strains were identified using a VITEK 2 (bioMérieux, Craponne, France) after confirmation by biochemical tests. For antibiotic susceptibility testing, minimum inhibitory concentration was determined using E-test strips (HiMedia, Mumbai, India) or VITEK 2 antibiotic susceptibility testing cards according to the manufacturer's protocol.19 Antibiotics tested included ciprofloxacin, moxifloxacin, gatifloxacin, ofloaxacin, ceftazidime, gentamicin, tetracycline, amikacin, tobramycin, piperacillin, norfloxacin, colistin, and imipenem. All results were compared to the Clinical and Laboratory Standards Institute interpretative guidelines, and the isolates were classified as susceptible or multidrug-resistant.34 (link) Multiple drug-resistant phenotypes were assigned for strains that were resistant to three or more classes of antibiotics.
E test strip
Manufactured by HiMedia
Sourced in India
E-test strips are a type of laboratory equipment used for antimicrobial susceptibility testing. They provide a quantitative measure of the minimum inhibitory concentration (MIC) of an antimicrobial agent against a specific microorganism.
Automatically generated - may contain errors
Lab products found in correlation
Vitek 2, by bioMérieux (1 mentions)
P aeruginosa atcc 27853 strain, by American Type Culture Collection (1 mentions)
Microscan, by Beckman Coulter (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Thermo Fisher Scientific (1 mentions)
Vancomycin, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
7 protocols using e test strip
1
Antibiotic Resistance in P. aeruginosa
2
Antibiotic Susceptibility Testing of Pseudomonas aeruginosa
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For antibiotic susceptibility testing, minimum inhibitory concentration (MIC) was determined using E-test strips (Himedia) or VITEK® 2 AST cards according to the manufacturer’s protocol [33 (link)] and this included ciprofloxacin, moxifloxacin, gatifloxacin, ofloaxacin, ceftazidime, gentamicin, tetracycline, amikacin, tobramycin, piperacillin, norfloxacin, colistin and imipenem. All results were compared to the Clinical and Laboratory Standards Institute (CLSI) interpretative guidelines and the isolates were classified as susceptible (S), susceptible dose dependent (SDD), and resistant (R) [34 (link)]. However, for the purpose of analysis, SDD isolates were clubbed with susceptible isolates (S-PA). Multiple drug resistant (MDR) phenotype was assigned for strains that was resistant to ≥3 classes of antibiotics. The P. aeruginosa ATCC 27853 strain (American Type Culture Collection), was used as the quality control. The P. aeruginosa phenotype was defined as MDR and XDR according to the international expert proposal for interim standards guidelines [18 (link)].
3
Candida Colonization Assessment Protocol
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Sputum, pharyngeal swab, umbilical swab, axillary swab, rectal swabs and urine were collected on day 1, day 4 and day 8 and followed up for each febrile case. All yeast isolates were subjected to Gram’s stain, germ tube test followed by identification on MICROSCAN (from Beckman Coulter). The yeast isolates were subjected to antifungal susceptibility testing using the commercial E-test strips (Hi-Media, Mumbai, India) for fluconazole (0.016–256 mcg/mL), Amphotericin-B (0.002–32 mcg/mL) and caspofungin (0.002–32 mcg/mL). Interpretive guidelines for in vitro susceptibility of Candida spp. (M27-S3 and S4) was followed to determine the susceptibility of isolates to different antifungals.
Based on colonization index on day 1, day 4 and day 8, candida colonization was evaluated among all febrile patients. The colonization index was defined as the ratio of the number of distinct body sites colonized by Candida spp. to the total number of body sites swabbed and cultured. A colonization index value ≥0.5 was considered as the threshold for significant candida colonization [5 (link)].
Based on colonization index on day 1, day 4 and day 8, candida colonization was evaluated among all febrile patients. The colonization index was defined as the ratio of the number of distinct body sites colonized by Candida spp. to the total number of body sites swabbed and cultured. A colonization index value ≥0.5 was considered as the threshold for significant candida colonization [5 (link)].
4
ESBL Detection: Ceftazidime and Clavulanic Acid
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The ceftazidime (30 μg) disc alone and in combination with clavulanic acid (Ceftazidime + Clavulanic acid, 30/10 μg disc) was applied onto a plate of Mueller Hinton Agar (MHA) for ESBL detection, which was inoculated with the test strain. The culture plates were incubated at 37 0 C for 16-18 hr and identified more than 5 mm zone of inhibition around the combination disc in comparison to the ceftazidime disc alone was deliberated to be a marker for ESBL production. e-test E-test was applied for further confirmation of ESBL isolates were made by evaluation of minimum inhibitory concentration (MIC). The MICs were evaluated by E-test strips (Hi-Media Laboratories Pvt. Limited, Mumbai). Two ends of The E-test strips were impregnated with cefotaxime (CTX) and cefotaxime + clavulanic acid (CTX+). The E-test strip was placed on the Mueller Hinton Agar (MHA) plate at centre and incubated aerobically at 37°C for 16-18 hours. The interpretation of MIC was considered as the value at the intersection of the growth ellipse with the strip. The ratio of the MIC value of cefotaxime to the MIC value of cefotaxime in combination with clavulanic acid was more than 8 or no zone was observed around cefotaxime but zone was obtained in cefotaxime and clavulanic acid combination was considered as an ESBL producer.
5
Tigecycline and Colistin MIC in CRE Isolates
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The minimum inhibitory concentration (MIC) of tigecycline and colistin susceptibility was determined for CRE isolates by using the E-test strips ranging from 0.016-256 μg /ml (Hi-Media, India) according to the manufacturer's instructions. Bacteria were cultured on a blood agar plate for 18h at 37°C and colonies resuspended in sterile saline to 0.5 McFarland standards. Each suspension was inoculated on a 90-mm diameter Mueller Hinton agar plate and E test strips were applied as recommended by the manufacturer. Results were recorded after 16-20h of incubation. Quality of media, and E test strips were checked with E. coli ATCC 25922.
The interpretation of tigecycline and colistin MIC was done by using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guideline for Enterobacteriaceae. The tigecycline MIC breakpoints were used as ≤1 and ≥2 mcg/ml for the susceptible and the resistant strains, respectively. For colistin, MIC ≤ 2 mcg/L was regarded as susceptible while ≥ 2 mcg/ml as resistant [15] .
The interpretation of tigecycline and colistin MIC was done by using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guideline for Enterobacteriaceae. The tigecycline MIC breakpoints were used as ≤1 and ≥2 mcg/ml for the susceptible and the resistant strains, respectively. For colistin, MIC ≤ 2 mcg/L was regarded as susceptible while ≥ 2 mcg/ml as resistant [15] .
6
Vancomycin Susceptibility Testing of S. aureus
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Carpet cultures of bacterial suspension were performed over the Mueller Hinton agar (MHA) plate. The E-test strip (HiMedia) was placed on an MHA plate with an MIC scale facing upward and the concentration maximum nearest to the rim of the plate. Then, the plates were incubated for 24 h at 37°C, and the zone of inhibition was measured [20 ]. The isolates were interpreted as vancomycin-sensitive S. aureus (VSSA), VISA, and VRSA if the size of zone of inhibition was ≥20 mm, 17–19 mm, and ≤16 mm, respectively. VRSA isolates were preserved in 20% glycerol containing tryptic soya broth and kept at −70°C until subsequent molecular tests were performed [21 (link)].
7
Antibiotic Susceptibility Profiling of Bifidobacteria
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Following the E-test protocol, a sterile cotton swab was first dipped in the inoculum (about 3×108 c.f.u. ml−1) and manually dispersed on the MRS/Bifido/TPY/MH agar plates. The plates were then allowed to dry for 15–30 min before the E-test strip was applied (Hi-Media Laboratories). The MIC values for ten different antibiotics, including inhibitors of protein synthesis (gentamycin, GEN10; tetracycline, TET30; chloramphenicol, CHL30; erythromycin, ERY15), inhibitors of cell wall synthesis (amoxicillin/amoxyclave, AMX10), and other widely used antibiotics such as ampicillin, tetracycline, kanamycin, pristomycin, streptomycin and vancomycin (obtained from Invitrogen), were determined for ten different bifidobacteria (Table 1 ).
The E-test (biodisk) method was followed to develop an antibiogram on different media such as Brucella agar, LSM agar supplemented with cysteine, TPY and MH agar. Susceptibility or resistance testing were performed according to the NCCLS [18 ]. The concentrations employed in the E-test were 0.016–256 g ml−1 for all antibiotics. Finally, the E-test agar plates underwent a 48 h anaerobic incubation in a gas jar at 37 °C (Hi-Media Laboratories; portable anaerobic workstation). Following incubation, the MICs were calculated and determined in triplicate for each strain.
The E-test (biodisk) method was followed to develop an antibiogram on different media such as Brucella agar, LSM agar supplemented with cysteine, TPY and MH agar. Susceptibility or resistance testing were performed according to the NCCLS [18 ]. The concentrations employed in the E-test were 0.016–256 g ml−1 for all antibiotics. Finally, the E-test agar plates underwent a 48 h anaerobic incubation in a gas jar at 37 °C (Hi-Media Laboratories; portable anaerobic workstation). Following incubation, the MICs were calculated and determined in triplicate for each strain.
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