Multi-drug resistant (MDR) P. aeruginosa strain PAO1 (ATCC 47085) and MDR E. coli O157:H7 strain SMS-3-5 (ATCC BAA-1743), both isolates from humans or the environment, and not produce associated, were revived from freezer stocks stored at -80°C by streaking onto Tryptic Soy Agar (TSA, Becton Dickinson, Franklin Lakes, NJ, United States) and incubating for 24 h at 37°C to obtain isolated colonies. An isolated colony of P. aeruginosa was streaked onto Pseudomonas Isolation Agar (PIA, Becton Dickinson, Franklin Lakes, NJ, United States) and an isolated colony of E. coli O157:H7 was streaked on to Eosin Methylene Blue Agar (EMB, Becton Dickinson, Franklin Lakes, NJ, United States) followed by incubation for 24 h at 37°C. Separate single colonies from PIA and EMB were incubated separately in Tryptic Soy Broth (TSB, Becton Dickinson, Franklin Lakes, NJ, United States) at 180 rpm for 24 h at 37°C. Cells were washed two times in 0.1% (wt/vol) peptone (Becton Dickinson, Franklin Lakes, NJ, United States) and were separately suspended in 9 ml of 0.1% (wt/vol) peptone to prepare the inoculation solution.
Eosin methylene blue agar
Manufactured by BD
Sourced in United States, Germany
Eosin methylene blue agar is a culture medium used for the isolation and differentiation of Gram-negative bacteria, particularly members of the Enterobacteriaceae family. It is a selective and differential medium that inhibits the growth of Gram-positive bacteria while promoting the growth of Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
E coli broth, by Thermo Fisher Scientific (2 mentions)
Macconkey agar, by Thermo Fisher Scientific (2 mentions)
Maldi tof, by bioMérieux (2 mentions)
Peptone, by BD (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Tryptic soy agar, by BD (1 mentions)
Pseudomonas isolation agar pia, by BD (1 mentions)
Blood agar, by BD (1 mentions)
Salmonella shigella agar, by BD (1 mentions)
Sabouraud dextrose agar, by BD (1 mentions)
Tissueruptor, by Qiagen (1 mentions)
Autoplate, by Advanced Instruments (1 mentions)
Macconkey agar plate, by BD (1 mentions)
Matrix assisted laser desorption ionization time of flight mass spectrometry, by bioMérieux (1 mentions)
Macconkey agar, by BD (1 mentions)
Blood agar, by Asan Pharmaceutical (1 mentions)
Phoenix 100, by BD (1 mentions)
Sheep blood agar, by BD (1 mentions)
14 protocols using eosin methylene blue agar
1
Reviving and Culturing MDR Pathogens
2
Bacterial Contamination Analysis of Contact Lens Cases
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Blood agar, chocolate agar, and eosin methylene blue agar (all from BBLTM, Becton Dickinson, Sparks, MD) were used as the standard culture system [20 (link)]. Ten microliters of the case fluid was then homogenously spread on each culture plate at room temperature. After incubation at 35 °C in an atmosphere containing 5% CO2 for 72 h, the bacteria obtained from the three culture media were identified with Gram staining, followed by standard biochemical tests for identification of bacteria [20 (link)]. The bioburden (CFU/ml) of each isolated strain was recorded for each case, which was used to characterize the degree of bacterial contamination in each lens case.
3
Microbial Contamination Evaluation in Food
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Determination of Salmonella and E. coli were followed according to Mexican legislation (NOM-210-SSA-2014) as an indicator of microorganisms of contamination, using Salmonella Shigella Agar (Becton Dickinson; Heidelberg, Germany) and Eosin Methylene Blue Agar (Becton Dickinson; Heidelberg, Germany), respectively. For the determination of molds and yeasts in food (Sabouraud Dextrose Agar, Becton Dickinson; Heidelberg, Germany), the methodology of NOM-11-SSA-1994 was followed. In addition, NOM-092-SSA-1994 was followed for the aerobic bacteria count (aerobic mesophiles) by using Standard Methods Agar (DIBICO; Cuautitlan Izcalli, Mexico). The samples were incubated in petri dishes (90 × 15 mm) for 24 h in an oven (FELISA model FE-131; Zapopan, Jalisco. Mexico) at 37 ± 2 °C. The results were expressed as the presence or absence of microorganisms that are indicative of an efficient pasteurization process.
4
Pathogen Adhesion Inhibition Assay
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The ability of L. brevis strains and prebiotic to inhibit the
adhesion of the pathogen to the HT-29 cell line was evaluated following the
method of Jang et al. (2019) (link). The
confluent HT-29 monolayers were inoculated with 1:1 mixture of pathogenic
bacteria and L. brevis (1×108 CFU/mL) and
incubated at 37°C for 2 h in a CO2 incubator. The prebiotics
(final concentration, 20 mg/mL) were added to each well immediately before
incubation. Samples without LAB or prebiotics were considered as control. To
determine the number (CFU/mL) of pathogens that adhered to the HT-29 cells, the
bacterial cells were harvested with 1% Triton X-100 and serially diluted
in PBS and plated on the Listeria selective agar and eosin methylene blue agar
(Becton-Dickinson, Franklin Lakes, NJ, USA) for detecting L.
monocytogenes and E. coli, respectively. The
plates were incubated at 37°C for 24 h.
adhesion of the pathogen to the HT-29 cell line was evaluated following the
method of Jang et al. (2019) (link). The
confluent HT-29 monolayers were inoculated with 1:1 mixture of pathogenic
bacteria and L. brevis (1×108 CFU/mL) and
incubated at 37°C for 2 h in a CO2 incubator. The prebiotics
(final concentration, 20 mg/mL) were added to each well immediately before
incubation. Samples without LAB or prebiotics were considered as control. To
determine the number (CFU/mL) of pathogens that adhered to the HT-29 cells, the
bacterial cells were harvested with 1% Triton X-100 and serially diluted
in PBS and plated on the Listeria selective agar and eosin methylene blue agar
(Becton-Dickinson, Franklin Lakes, NJ, USA) for detecting L.
monocytogenes and E. coli, respectively. The
plates were incubated at 37°C for 24 h.
5
Biopsy Tissue Sample Analysis Protocol
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Three replicate 4 mm biopsy punch tissue samples were evaluated per wound. Three wounds were evaluated per pig on days 2, 4, 7, and 10 postinoculation. The sample was placed in 1 mL of sterile PBS in a 14-mL conical tube and homogenized (TissueRuptor; Qiagen Sciences, Inc., Germantown, MD). Serial 10-fold dilutions of homogenate were plated via spiral plater onto eosin methylene blue agar (Becton, Dickinson and Co). Plates were incubated overnight at 37°C, and then, CFU were enumerated.
6
Quantifying Wound Bacterial Burden
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To examine CFU burden within the wound bed, mice were euthanized with ketamine (250 mg/kg) and xylazine (25 mg/kg) overdose according to protocol on day 1 or day 3. A 4-mm disposable skin biopsy punch (Acuderm Inc., Fort Lauderdale, FL) was then used to sample a disc from the wound bed. The sample was manually disrupted in PBS, and serial 10-fold dilutions were plated via a spiral plater (Autoplate; Advanced Instruments, Inc., Norwood, MA) on eosin methylene blue agar (Becton, Dickinson and Co., Sparks, MD). Plates were incubated overnight at 37°C and then enumerated.
Separately, the CFU was determined at the 4-h postinoculation time point. The dorsal wounds were removed en bloc by severing the cervical and lumbar spinal column and trimming the tissue at >2 cm beyond the wound edge. The tissue was processed using a sterile homogenizer, and serial 10-fold dilutions were plated via a spiral plater and enumerated as above.
Separately, the CFU was determined at the 4-h postinoculation time point. The dorsal wounds were removed en bloc by severing the cervical and lumbar spinal column and trimming the tissue at >2 cm beyond the wound edge. The tissue was processed using a sterile homogenizer, and serial 10-fold dilutions were plated via a spiral plater and enumerated as above.
7
Quantifying Bacterial Burden in Tissues
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After euthanasia, wound bed, spleen, liver, and lung were aseptically collected to quantify viable bacteria. For surviving mice on day 7, a cotton swab, padded on the wound surface suspended in sterile PBS, was used for bacterial count. After PBS rinsing, tissues were weighed and then manually ground using a syringe plunger. Diluted homogenate was plated onto eosin methylene blue agar (Becton, Dickinson and Co., Sparks, Maryland) and incubated at 37 • C overnight. CFU count was expressed as CFU/g tissue.
8
Monitoring Antibiotic Resistance in Wild Birds
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To isolate E. coli for monitoring antibiotic resistance in wild birds during rehabilitation, fecal swabs from 17 wild birds in the wild state and release state were inoculated into 2 mL of E. coli broth (Oxoid, Basingstoke, UK) and enriched overnight at 37°C. After enrichment, 100 μL of the culture broth was spread on MacConkey agar (Oxoid) and incubated at 37°C for 24 h. Cultures were then streaked on eosin methylene blue agar (BD, Sparks, MD, USA), and colonies exhibiting the culture characteristics of E. coli were pure cultured and confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry. As a result, E. coli was isolated from 15 birds and further analyzed for antibiotic susceptibility.
Disk diffusion susceptibility tests (Kirby-Bauer method) were conducted for eight antibiotics: amoxicillin-clavulanic acid (20/10 μg), ampicillin (10 μg), cefotaxime (30 μg), imipenem (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), colistin (10 μg), and cefoxitin (30 μg). Antibiotic susceptibility results were interpreted following the Clinical and Laboratory Standards Institute guidelines.
Disk diffusion susceptibility tests (Kirby-Bauer method) were conducted for eight antibiotics: amoxicillin-clavulanic acid (20/10 μg), ampicillin (10 μg), cefotaxime (30 μg), imipenem (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), colistin (10 μg), and cefoxitin (30 μg). Antibiotic susceptibility results were interpreted following the Clinical and Laboratory Standards Institute guidelines.
9
Monitoring Antibiotic Resistance in Wild Birds
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To isolate E. coli for monitoring antibiotic resistance in wild birds during rehabilitation, fecal swabs from 17 wild birds in the wild state and release state were inoculated into 2 mL of E. coli broth (Oxoid, Basingstoke, UK) and enriched overnight at 37°C. After enrichment, 100 μL of the culture broth was spread on MacConkey agar (Oxoid) and incubated at 37°C for 24 h. Cultures were then streaked on eosin methylene blue agar (BD, Sparks, MD, USA), and colonies exhibiting the culture characteristics of E. coli were pure cultured and confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry. As a result, E. coli was isolated from 15 birds and further analyzed for antibiotic susceptibility.
Disk diffusion susceptibility tests (Kirby-Bauer method) were conducted for eight antibiotics: amoxicillin-clavulanic acid (20/10 μg), ampicillin (10 μg), cefotaxime (30 μg), imipenem (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), colistin (10 μg), and cefoxitin (30 μg). Antibiotic susceptibility results were interpreted following the Clinical and Laboratory Standards Institute guidelines.
Disk diffusion susceptibility tests (Kirby-Bauer method) were conducted for eight antibiotics: amoxicillin-clavulanic acid (20/10 μg), ampicillin (10 μg), cefotaxime (30 μg), imipenem (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), colistin (10 μg), and cefoxitin (30 μg). Antibiotic susceptibility results were interpreted following the Clinical and Laboratory Standards Institute guidelines.
10
Antimicrobial Resistance Profiling of E. coli
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Samples were processed, and E. coli was isolated as described previously [29 (link)] using eosin methylene blue agar (BD, Sparks, MD, USA) and MacConkey agar plates (BD). Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (bioMérieux, Marcy l'Étoile, France).
Antimicrobial susceptibility was assessed by determining the minimum inhibitory concentrations (MICs) for 16 antimicrobial agents using the broth microdilution method with a commercially available Sensititre® panel KRVP4F (TREK Diagnostic Systems, West Sussex, UK) according to the manufacturer's instructions. The following antibiotics were tested: ampicillin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur, ceftazidime, cefepime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. The reference strain E. coli ATCC 25922 was used as quality control when determining MICs. The MIC was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). When CLSI breakpoints were not available, the MIC was interpreted according to the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP, 2014). Multidrug resistance was defined as resistance to ≥3 antibiotic subclasses.
Antimicrobial susceptibility was assessed by determining the minimum inhibitory concentrations (MICs) for 16 antimicrobial agents using the broth microdilution method with a commercially available Sensititre® panel KRVP4F (TREK Diagnostic Systems, West Sussex, UK) according to the manufacturer's instructions. The following antibiotics were tested: ampicillin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur, ceftazidime, cefepime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. The reference strain E. coli ATCC 25922 was used as quality control when determining MICs. The MIC was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). When CLSI breakpoints were not available, the MIC was interpreted according to the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP, 2014). Multidrug resistance was defined as resistance to ≥3 antibiotic subclasses.
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