Tryptic soy agar medium was used to culture all tested bacterial strain. A 5000 μg/mL solution of EOs was prepared in dimethyl sulfoxide (DMSO), and twofold dilution in 100 μL of cation-adjusted Mueller Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO, USA) was added to the top well of a 96-well microdilution plate. The prepared stock solution of EOs was then serially twofold diluted in fresh CAMHB to obtain final concentrations of 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, and 19.5 μg/mL. The freshly harvested bacteria with approximately 1.5 × 108 CFU/mL final concentration were added to each well of 96-well microdilution plates and were incubated at 37 °C for 24 h. Gentamicin (Sigma-Aldrich, St. Louis, MO, USA) and DMSO were used as positive and negative controls, respectively [56 (link)]. Seven microorganisms were used to evaluate the antibacterial activities of C. winterianus (leaves and root) EOs: Five Gram-positive bacteria, Bacillus cereus (ATCC-14579), Staphylococcus epidermidis (ATCC-12228), Propionibacterium acnes (ATCC-11827), Staphylococcus aureus (ATCC-29213), and Streptococcus pyogenes (ATCC-19615) and two Gram-negative bacteria, Serratia marcescens (ATCC-14756) and Pseudomonas aeruginosa (ATCC-27853), using the microbroth dilution technique.
Camhb
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
CAMHB is a type of laboratory growth medium used for culturing microorganisms. It is a complex medium that provides the necessary nutrients and growth factors for the cultivation of a variety of bacteria. CAMHB is commonly used in antimicrobial susceptibility testing to determine the sensitivity of bacteria to different antibiotics.
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Lab products found in correlation
Camhb, by BD (4 mentions)
Tobramycin, by Merck Group (3 mentions)
Vancomycin, by Merck Group (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
Gentamicin, by Merck Group (2 mentions)
Glucose 6 phosphate (g6p), by Merck Group (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Trypticase soy agar, by bioMérieux (1 mentions)
Tigecycline, by Pfizer (1 mentions)
Mueller hinton agar (mha), by Solabia (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Gentamicin sulfate, by Merck Group (1 mentions)
Colistin, by Merck Group (1 mentions)
Probenecid, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Carbenicillin, by Research Products International (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Vancomycin powder, by Merck Group (1 mentions)
Col sulfate, by Merck Group (1 mentions)
Pentamidine, by MedChemExpress (1 mentions)
52 protocols using camhb
1
Antibacterial Efficacy of Cymbopogon winterianus EOs
2
Antibiotic Resistance Profiling of Acinetobacter baumannii
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An initial screen of antibiotic susceptibilities was done using a drop test, where 10 µL of different antibiotics were spotted onto a lawn of A. baumannii on LBA. To determine if the three A. baumannii host strains were multi, extensively or pan drug resistant, the more quantitative disc diffusion method was carried out on selected clinically relevant antibiotics according to CLSI guidelines [49 ]. A further investigation into the colistin, apramycin, tigecycline, and meropenem resistance profiles of the A. baumannii host strains, was performed according to broth microdilution methods outlined by EUCAST [50 (link)] using cation adjusted Mueller Hinton broth 2 (CA-MHB) (Sigma-Aldrich—17.5 g/L acid hydrolysate of casein, 3 g/L beef extract, 1.5 g/L starch) and an initial inoculum of 5 × 105 CFU/mL. The CA-MHB used with tigecycline was either used fresh (<12 h) or frozen and used later, in order to optimise the activity of this antibiotic, which can be negatively impacted by media that has been stored for too long [10 (link)].
3
Antibacterial Activity of Wintergreen Oil
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Seven microorganisms were used to evaluate the antibacterial activities of some selected wintergreen essential oils: five Gram-positive bacteria, Bacillus cereus (ATCC-14579), Staphylococcus epidermidis (ATCC-12228), Propionibacterium acnes (ATCC-11827), Staphylococcus aureus (ATCC-29213), and Streptococcus pyogenes (ATCC-19615), and two Gram-negative bacteria, Serratia marcescens (ATCC-14756) and Pseudomonas aeruginosa (ATCC-27853), using the microbroth dilution technique. Tryptic soy agar medium was used to culture all tested bacterial strains. A 5000 μg/mL solution of wintergreen essential oil was prepared in dimethyl sulfoxide (DMSO) and twofold dilution in 100 μL of cation-adjusted Mueller Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO, USA) was added to the top well of a 96-well microdilution plate. The prepared stock solution of essential oils was then serially two-fold diluted in fresh CAMHB to obtain final concentrations of 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, and 19.5 μg/mL. The freshly harvested bacteria with approximately 1.5 × 108 CFU/mL final concentration were added to each well of 96-well microdilution plates and were incubated at 37 °C for 24 h. Gentamicin (Sigma-Aldrich, St. Louis, MO, USA) and DMSO were used as positive and negative controls, respectively [15 (link),19 (link)].
4
Antibiotic Susceptibility Testing Protocol
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CAMHB (Sigma–Aldrich) was used as the primary medium in all experiments. Where fosfomycin was used, the CAMHB was supplemented with 25 mg/L glucose-6-phosphate (G6P) (Sigma–Aldrich). Mueller–Hinton agar (MHA) was used in all agar plates. For drug-containing plates, MHA was supplemented with antibiotic (with an additional 25 mg/L G6P, if fosfomycin) to a concentration of four times the antibiotic MIC for the specific bacterial strain, prepared within each antibiotic’s stability time limits.
5
Urine and Iron-depleted Medium Effects on MIC
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Urine: midstream urine was obtained from young, male, healthy volunteers, frozen, thawed, pooled, sterile filtered (2 µm) and frozen again until usage to guarantee the usage of one single pooled batch of urine for all tests. The iron content of pooled urine, as determined in the central laboratory of the General Hospital of Vienna, was ∼ 0.05 mg/L/24 h.
CAMHB: CAMHB (Sigma–Aldrich, Steinheim, Germany) containing 17.5 g/L casein acid hydrolysate, 3 g/L beef extract and 1.5 g/L starch, 20–25 mg/L calcium, 10–12.5 mg/L magnesium and iron content ∼0.05 mg/L served as an additional reference medium for MIC testing as iron concentrations are similar to the physiological concentrations found in urine.
ID-CAMHB: the broth was produced as recommended by the EUCAST guidance document on cefiderocol BMD (latest version, December 2020) and as described in detail by Hackel et al.14 (link) In short, ID-CAMHB was prepared by adding 100 g of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) to 1 L of freshly prepared CAMHB. The suspension was stirred for 2 h at room temperature (22°C), filtered using a 0.2 μm filter and cation adjusted as recommended.
The pH was set for all tested media (pooled urine, CAMHB and ID-CAMHB) with HCl or NaOH directly before the start of the experiments to values of 5, 7 and 8.
CAMHB: CAMHB (Sigma–Aldrich, Steinheim, Germany) containing 17.5 g/L casein acid hydrolysate, 3 g/L beef extract and 1.5 g/L starch, 20–25 mg/L calcium, 10–12.5 mg/L magnesium and iron content ∼0.05 mg/L served as an additional reference medium for MIC testing as iron concentrations are similar to the physiological concentrations found in urine.
ID-CAMHB: the broth was produced as recommended by the EUCAST guidance document on cefiderocol BMD (latest version, December 2020) and as described in detail by Hackel et al.14 (link) In short, ID-CAMHB was prepared by adding 100 g of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) to 1 L of freshly prepared CAMHB. The suspension was stirred for 2 h at room temperature (22°C), filtered using a 0.2 μm filter and cation adjusted as recommended.
The pH was set for all tested media (pooled urine, CAMHB and ID-CAMHB) with HCl or NaOH directly before the start of the experiments to values of 5, 7 and 8.
6
Antimicrobial Susceptibility Testing Protocol
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Initial AST for cefpodoxime (CPD), cefoxitin (FOX), TZP, meropenem (MEM), CIP, cefotetan (CTT), amikacin (AMK), ertapenem (ETP), AMC, CHL and ampicillin (AMP) was performed in RLUH clinical laboratory using the disk diffusion method, according to either British Society for Antimicrobial Chemotherapy (BSAC) or EUCAST guidelines for Antimicrobial Susceptibility Testing41 –43 .
MIC for TZP, GEN, CIP, CHL, AMC and TET were performed using the broth microdilution method, in cation adjusted Mueller Hinton Broth (CA-MHB, Sigma, UK), following EUCAST Guidelines44 . Efflux pump inhibition was performed using PAβN as a supplement in CA-MHB at a final concentration of 50 µM, inhibition of TEM-1B was determined with piperacillin with increasing concentrations of tazobactam (4, 8 and 16 µg/ml) and inhibition of OXA-1 was determined with TZP plus 100 mM sodium chloride (Sigma, UK). Each MIC was performed in triplicate using independent bacterial cultures, each with three technical replicates.
MIC for TZP, GEN, CIP, CHL, AMC and TET were performed using the broth microdilution method, in cation adjusted Mueller Hinton Broth (CA-MHB, Sigma, UK), following EUCAST Guidelines44 . Efflux pump inhibition was performed using PAβN as a supplement in CA-MHB at a final concentration of 50 µM, inhibition of TEM-1B was determined with piperacillin with increasing concentrations of tazobactam (4, 8 and 16 µg/ml) and inhibition of OXA-1 was determined with TZP plus 100 mM sodium chloride (Sigma, UK). Each MIC was performed in triplicate using independent bacterial cultures, each with three technical replicates.
7
Microdilution Assay for Antimicrobial Susceptibility
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According to the recommendations of the CLSI, the broth microdilution method was used to determine the MIC values of QUE and antibiotics [47 ]. Cation-adjusted Mueller Hinton Broth (MHB) (CAMHB, Merck KGaA, Darmstadt, Germany) was used in 96-well microplates to prepare the double serial dilutions of antimicrobial agents. The ranges for QUE and antibiotic concentrations were 0.25–512 and 0.0625–128 µg/mL, respectively. The bacterial suspension was added to each well after being adjusted to the 0.5 McFarland standard (final bacterial concentration: 5 × 105 colony forming units [CFU]/mL). Each microdilution plate also included controls for bacterial growth (CAMHB + bacteria) and medium sterility (CAMHB). Microdilution plates were incubated at 35 ± 2 °C for 18–24 h. The MIC values were calculated by comparing the growth density in the antibiotic-containing wells to that in the control wells used in each test set. Each experiment was conducted in triplicate.
8
Determination of AEEP and Q Antimicrobial Activity
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A broth microdilution method was used to determine MIC according to CLSI broth microdilution method [24 ]. First, bacterial suspension (105 CFU ml− 1) was inoculated into CAMHB (Merck, Germany) and dispensed at in 96-well (0.2 ml) microtiter plates. Two-fold serial dilutions of AEEP and Q were prepared and transferred to each well of the original extract in DMSO with final concentration from 1.56 to 1000 μg/ml. Final concentration of DMSO as solvent was 1%. The number of wells in each plate were allocated to negative control, a sterility control, and a control for the solvent (DMSO), the antibiotic penicillin tested as positive controls. Plates were anaerobically incubated for 24 h at 37 °C. MIC was determined as the lowest concentration of samples that had no macroscopically visible growth. Minimum bactericidal concentration (MBC) was determined by sub-culturing of three previous wells on Mueller Hinton agar and incubating for 24 h. The lowest concentration of samples with no bacterial growth (99% inhibition) was reported as MBC.
9
Bacterial Isolate Inoculation and Dilution
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The isolates were inoculated on a TSA medium (Trypticase soy agar, BioMérieux, Marcy-l’Étoile, France) at 37 °C for 18–24 h. The strains were suspended at 108 CFU/mL in 0.9% NaCl and diluted 1/100 (106 CFU/mL) in cation adjusted Mueller–Hinton broth media (CAMHB, Merck KGaA, Darmstadt, Germany).
10
Broth Microdilution for Oxacillin and Carvacrol MIC
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Broth microdilution method was used to detect MIC values of oxacillin and carvacrol (14) . 50 µl of antimicrobial agent was added to the first wells of 96well microdilution plates which contains 50 µl of cationadjusted Mueller Hinton Broth (CAMHB, Merck KGaA, Darmstadt, Germany). Serial dilutions were then performed. The concentration range of oxacillin was 128-0.0625 µg/ml, whereas the range of carvacrol was 512-0.25 µg/ml. 50 µl of bacterial suspension was added to each well (5x10 5 cfu/ml). In addition, bacterial growth control (CAMHB+bacteria) and medium sterility control (CAMHB) for each microdilution plate were studied. The microdilution plates were incubated at 35±2°C for 16-20 hours in an incubator. MIC values were determined by comparing the growth density in the wells containing antibiotics with the growth density in the control wells (without antibiotics). MIC is the lowest antimicrobial drug concentration that completely inhibits the growth of bacteria in microdilution wells and can be determined by the naked eye. Each experiment was repeated three times.
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