A nonpathogenic E. coli isolate (ESBL10682, isolated from the excreta of one day old broilers within the RESET program), harboring the blaCTX-M-1 gene and belonging to the B1 subgroup, was chosen as the donor. The strain Salmonella Typhimurium L1219-R32 (isolated from pigs) was chosen as the recipient strain. This mating pair was revealed to be the best fit for the study design in a previous study obtaining conjugation kinetics for five potential E. coli donor strains and six potential Enterobacteriaceae recipients every 2 h for 22 h [38 ]. From these results, the mating pair was known to result in a conjugation frequency of approximately 105 transconjugants/donor after 4 h of co-incubation (donor:recipient 1:1; 105 cells/mL starting conditions) [38 ]. All cultures were obtained from cryo-stocks and cultured in Mueller Hinton 2 broth (Sigma-Aldrich, Chemie GmbH, Darmstadt, Germany). Culturing of the strains was done in Mueller Hinton 2 broth, supplemented with 8 µg/mL cefotaxime (CTX) (Alfa Aesar, Thermo Fisher GmbH, Schwerte, Germany) for E. coli incubation or 300 µg/mL sulfamethoxazole/trimethoprim (SXT) (Sigma-Aldrich, Chemie GmbH, Darmstadt, Germany) for the Salmonella Typhimurium strain. All strains were incubated aerobically at 37 °C.
Mueller hinton broth 2
Manufactured by Merck Group
Sourced in United States, Germany
Mueller Hinton Broth 2 is a microbiological culture medium used for the growth and isolation of a variety of bacterial species. It provides the necessary nutrients and growth factors to support the cultivation of various microorganisms, including those involved in antimicrobial susceptibility testing.
Automatically generated - may contain errors
Lab products found in correlation
Cellstain double staining kit, by Merck Group (1 mentions)
Caspase 3 7 assay, by Promega (1 mentions)
Dichlobenil, by Merck Group (1 mentions)
Bifenox, by Merck Group (1 mentions)
Gsh gssg glo assay kit, by Promega (1 mentions)
Folin ciocalteu reagent, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Penicillin, by PAN Biotech (1 mentions)
Rpmi 1640 medium, by PAN Biotech (1 mentions)
Streptomycin, by PAN Biotech (1 mentions)
Trypsin, by PAN Biotech (1 mentions)
Phosphate buffered saline (pbs), by PAN Biotech (1 mentions)
Cefotaxime, by Merck Group (1 mentions)
Ciprofloxacin, by Biosynth (1 mentions)
Doxycycline, by Biosynth (1 mentions)
Amoxicillin, by Merck Group (1 mentions)
Amikacin, by Merck Group (1 mentions)
21 protocols using mueller hinton broth 2
1
Conjugation Kinetics of ESBL-Producing E. coli
2
Conjugative Transfer of ESBL-Producing E. coli in Vitro
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experimental design comprised the ESBL-producing donor strain Escherichia coli ESBL10682, derived from broiler excreta within the RESET program [26 ]. This strain belonged to the phylogenic group B1 and produced the enzyme CTX-M-1. Furthermore, the strain Salmonella Typhimurium L1219-R32 served as the recipient. Susceptibility of donor and recipient against various antibiotics was investigated by disc diffusion test (Table S1 Saliu et al., manuscript submitted). This conjugative pair was known to transfer plasmids in vitro at a conjugation frequency (CF) of 10−4–10−5 when incubated in Mueller Hinton 2 Broth (Sigma-Aldrich, Chemie GmbH, Germany) for 4 h [27 (link)]. All samples were cultivated aerobically at 37 °C for 4 h.
3
Time-Killing Assay for Antibacterial Agents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Classical time-killing assays were performed according to the American Society for Microbiology guidelines [26 ]. In brief, bacteria exponentially grown at 37°C, 150 rpm, in Mueller-Hinton II broth (Sigma-Aldrich, Germany) were diluted with fresh medium to obtain an OD600nm of 0.015 (approximately 5.107 cells/mL). Thirty milliliters were then evenly aliquoted in 5 Erlenmeyer flasks corresponding to the 5 different conditions: a culture without antibacterial agents; cultures with antibiotics 1, 2, and 3; and a culture with phages. At time 0 (T0), antibacterial agents were added and each flask was sampled at 15, 30, 45, 60, and 180 minutes. The control (without antibacterial agents) was also sampled at T0. The bacterial titer (in triplicate), the instantaneous bacterial viability (in triplicate), and the free endotoxin level (in duplicate) were determined. The detection threshold for the count of colony-forming units (CFUs) is 1 × 102 CFU/mL. Killing assays (and subsequent analysis) were independently repeated twice for each strain.
Microscope observations were performed on samples taken at 180 minutes with the remaining volume of culture. After centrifugation (7000g, 5 minutes), the pellet was resuspended in 100 µL of phosphate-buffered saline and 5 µL was dropped on a glass slide to be observed with a phase contrast microscope.
Microscope observations were performed on samples taken at 180 minutes with the remaining volume of culture. After centrifugation (7000g, 5 minutes), the pellet was resuspended in 100 µL of phosphate-buffered saline and 5 µL was dropped on a glass slide to be observed with a phase contrast microscope.
4
Cytotoxicity Evaluation of Pesticides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI-1640 medium, containing glucose at 4.5 mg/mL (25 mM), penicillin, streptomycin, trypsin–EDTA, FBS and PBS (without Ca and Mg, pH 7.4) were provided by PAN Biotech. GSH/GSSG-Glo™ Assay kit, Caspase 3/7 Assay and Caspase 9 Assay were provided by Promega Madison, WI. SDS, TCA, TBA, Folin-Ciocalteu reagent were provided by Sigma-Aldrich and DTNB by Serva. Dichlorodihydrofluorescein diacetate assay (DCFH-DA). Bifenox and Dichlobenil and Mueller Hinton II Broth were provided by Sigma-Aldrich, St. Louis, MO, USA. Cell stain double staining kit containing propionium iodide and calcein—AM was provided by Sigma-Aldrich, St. Louis, MO, USA. MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich and fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I from BD Pharmingen (San Diego, CA, USA).
5
Determining Minimal Inhibitory Concentrations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Minimal inhibitory concentrations (MICs) for LytM_EAD, Lss, and Chimera (G391A, G392A) were determined as previously described39 (link) by growing S. aureus NCTC 8325-4 in 96-well plate (4 × 105 CFU/ml) in 100 μl of Mueller Hinton II Broth (Sigma-Aldrich) supplemented with 2% NaCl and 0.1% bovine serum albumin (BSA). Enzymes were added at concentrations ranged from 0.25 to 0.00025 μg/ml in twofold dilutions in the media. A positive control without added enzymes was included in each assay. Plates were incubated at 37°C with shaking (150 rpm) for 24 hr. The absorbance using microplate reader was measured at 595 nm and MIC values determined based on wells with no measurable bacterial growth.
6
EUCAST-Guided Antimicrobial Susceptibility Testing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The guidelines provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; https://www.eucast.org ) were followed for completion of MIC assays by the broth microdilution method. All MIC assays were conducted in cation-adjusted Mueller–Hinton II broth (Sigma-Aldrich, MO, USA) in 96-well round-bottom polypropylene microplates (Corning, NY, USA) and results determined visually following 18±2 h static incubation at 37 °C. Antibiotics were purchased from Melford Laboratories (amikacin, amoxicillin, amoxicillin-clavulanate, cefotaxime, colistin), Sigma-Aldrich (ceftazidime, ciprofloxacin, doxycycline, erythromycin, meropenem), Carbosynth (imipenem, relebactam), Fisher Scientific (cefepime) and Lonza (gentamicin). Antibiotic stocks were prepared in sterile deionized water, except erythromycin, which was prepared in dimethyl sulfoxide, and stored at −20 °C until use. A 16 : 1 ratio of amoxicillin to relebactam was used, as previously reported [52 (link)]. Classification of the isolate’s susceptibility to antibiotics was determined based on EUCAST breakpoints [53 ] using entries for Pseudomonas
7
Antimicrobial Peptide Efficacy Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reference strains were supplied by the Polish Collection of Microorganisms (Polish Academy of Sciences, Institute of Immunology and Experimental Therapy, Wroclaw, Poland). Minimal inhibitory concentration (MIC) was determined by the microbroth dilution method outlined by the National Committee for Clinical Laboratory Standards (2003 ). The following microbial strains were tested: Gram-positive: Rhodococcus equi ATCC 6939, Staphylococcus epidermidis, S. aureus ATCC 6538, Streptococcus pyogenes and Enterococcus faecalis; Gram-negative: E. coli ATCC 8739, Pseudomonas aeruginosa and fungi: C. albicans ATCC10231. Bacteria were suspended in Mueller–Hinton II broth (Sigma-Aldrich) at initial inocula of 5 × 105 cfu ( colony-forming units)/mL, while the Sabouraud 5 % dextrose broth of a pH of 7.4 (Sigma-Aldrich) was used for fungi (at initial inoculum of 5 × 103 cfu/mL). Microbial cells in polystyrene 96-well plates were exposed to the peptides at adequate concentrations (range 1–512 μg/mL) for 18 h at 37 °C (bacterial strains) or for 48 h at 25 °C (fungi). MIC was taken as the lowest drug concentration at which observable growth was inhibited. Experiments were performed in triplicate on three different days.
8
Meropenem Resistance Development in K. pneumoniae
Check if the same lab product or an alternative is used in the 5 most similar protocols
K. pneumoniae K66-45 was passaged in a 96-well microtiter plate with a consistent concentration of ZN148 and increasing concentrations of meropenem. To start the passaging, cation-adjusted Mueller-Hinton broth II medium (Sigma-Aldrich) containing 0.0625 mg/liter (0.5× MIC) and 50 or 100 μM ZN148 was inoculated with 1% of overnight culture, which was grown without selection. Six biological replicates were passaged in parallel, and from each of the six overnight cultures four wells were inoculated, resulting in 24 subcultures for each ZN148 concentration. After 24 h of incubation at 37°C and shaking at 180 rpm, the wells were visually checked for growth. Passaging was continued with all subcultures showing growth by transferring 1 μl of subculture to 100 μl of fresh medium with 0.125 mg/liter meropenem (1× MIC) and 50 or 100 μM ZN148. This process was continued by increasing the meropenem concentration by 2-fold every 24 h until none of the subcultures showed growth.
+ Expand
9
Screening and Colistin Susceptibility of mcr Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All isolates were screened by a multiplex PCR to detect five mcr genes (mcr-1 to mcr-5) as described previously58 (link). E. coli NCTC 13846, E. coli KP3711 (link), E. coli 2013-SQ35258 (link), E. coli DH5α13 (link), and Salmonella 13-SA0171814 (link) were used as the positive controls for mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5, respectively. Gene Ruler 100 bp Plus DNA Ladder (Thermo Fisher Scientific, USA) was used as an external reference control. All the mcr-positive E. coli strains were subjected to MIC determination. The MIC for colistin was determined by the broth microdilution (BMD) method according to ISO-standard (20776-1)59 . Colistin sulphate (Sigma-Aldrich, Saint Louis, MO, USA) and cation-adjusted Mueller–Hinton Broth II (Sigma-Aldrich, St Louis, MO, USA) were used in the BMD test. The quality control of the experiment was monitored by a resistant strain of E. coli NCTC 13846 (mcr-1-positive) and a susceptible strain of E. coli ATCC 25922. To test the colistin susceptibility of the mcr-1-negative isolates, all the isolates were streaked on the MacConkey agar with 2 mg/L Colistin sulphate. Growth was scored after overnight incubation of agar plates at 37˚C. The mcr-1-negative isolates which showed growth at this concentration were subjected to MIC determination for colistin as above.
10
Colistin Resistance Profiling in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the presumptive colistin-resistant E. coli (n = 105) colonies isolated from colistin-containing MacConkey agar plates were subjected to the broth microdilution (BMD) method to determine the MIC of colistin sulfate [30 ]. To standardize the method, a cation-adjusted Mueller–Hinton Broth II (Sigma-Aldrich, St Louis, MO, USA) was used. E. coli NCTC 13846 (colistin resistant) and E. coli ATCC 25922 (colistin susceptible) were used in all MIC experiments as the quality control strains.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!