Specimens including rectal swabs were collected from patients during the study period. Rectal swabs were plated on primary CHROMagar KPC medium (CHROMagar, Paris, France) for direct screening of CRE. Other clinical specimens except rectal swabs were plated on blood agar (Becton-Dickinson, Sparks, MD, USA) and MacConkey agar (Becton-Dickinson). The agar plates were incubated at 36.5 °C in ambient air for 16–24 h. Bacterial identification was done with VITEK® 2 system (bioMerieux, Marcy I’Etoile, France). Susceptibility testing to antibiotics including carbapenem was performed using the VITEK® 2 system according to the Clinical and Laboratory Standards Institute M100S guidelines [19 ]. Isolates were tested further to detect carbapenemase production using the modified Hodge test and carbapenemase inhibition test [20 (link)]. In addition, we performed a polymerase chain reaction assay targeting the genes encoding blaKPC, blaNDM, blaIMP,blaVIM, blaGES, and blaOXA-48 to confirm carbapenemase [21 (link)].
Blood agar
Manufactured by BD
Sourced in United States, Germany
Blood agar is a type of microbiological culture medium used for the isolation and identification of various microorganisms, particularly those that require or tolerate the presence of blood. It is composed of a standard nutrient agar base supplemented with defibrinated blood, typically 5-10% sheep or horse blood. The blood agar provides additional growth factors and nutrients to support the growth of fastidious bacteria and allows for the observation of hemolytic reactions, which can be a useful diagnostic characteristic.
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Lab products found in correlation
Macconkey agar, by BD (7 mentions)
Vitek 2 system, by bioMérieux (3 mentions)
Chocolate agar, by BD (3 mentions)
Kpc medium, by CHROMagar (2 mentions)
Scan 500, by Interscience (2 mentions)
Allprotect reagent, by Qiagen (2 mentions)
Trypticase soy broth (tsb), by BD (1 mentions)
Isovitalex, by BD (1 mentions)
Maldi tof, by Bruker (1 mentions)
Etest, by bioMérieux (1 mentions)
Eosin methylene blue agar, by BD (1 mentions)
Mcconkey agar, by BD (1 mentions)
Nutrient agar, by BD (1 mentions)
Mueller hinton broth and agar, by BD (1 mentions)
Live dead baclight bacterial viability and counting kit, by Thermo Fisher Scientific (1 mentions)
Catalase, by bioMérieux (1 mentions)
Mrs broth, by Solabia (1 mentions)
Api 50 ch gallery, by bioMérieux (1 mentions)
Mrs agar, by Solabia (1 mentions)
Api 50 chl medium, by bioMérieux (1 mentions)
35 protocols using blood agar
1
Screening and Identification of Carbapenem-Resistant Enterobacteriaceae
2
Characterization and Storage of Bacterial Isolates
3
Neisseria Strain Isolation and Preparation
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Gonoccocal and non-gonoccocal clinical and reference Neisseria strains were inoculated on chocolate agar with IsoVitaleX or blood agar (BectonDickinson, Sparks, United States (US)) and incubated over night at 37 °C in 5% CO2. Morphology of the colonies was checked and a single colony was subcultured before DNA isolation. Bacterial suspensions were prepared in sterile saline solution from two or three colonies (depending on the size of the colonies) picked with a pre-wetted sterile swab. The bacteria were pelleted by centrifugation at 2,000 g for 5 min and washed once. The pellet was resuspended in 500 µL MQ and boiled for 10 min. Cell debris was pelleted by centrifugation at 8,000 g for 2 min and the supernatant was stored at −20 °C.
4
Carbapenem-Resistant Enterobacteriaceae Screening
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Specimens including rectal swabs were collected from patients during the study period. Rectal swabs were plated on primary CHROMagar KPC medium (CHROMagar, Paris, France) for CRE screening. Other clinical specimens such as sputum, urine, and blood were plated on blood agar (Becton-Dickinson, Sparks, MD, USA) and MacConkey agar (Becton-Dickinson). Agar plates were incubated at 36.5 °C for 16–24 h. Taxonomic identification and antibiotic susceptibility tests, including carbapenem for isolates, were performed using the VITEK® 2 system (bioMerieux, Marcy I’Etoile, France) according to the Clinical and Laboratory Standards Institute M100S guidelines [18 ]. Carbapenemase production in each isolate was analyzed using the CarbaNP test [19 (link)]. To validate carbapenamase production in isolates, target genes encoding blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48 were amplified and confirmed by gel electrophoresis [20 (link)].
5
Vancomycin-resistant Enterococcus identification
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We collected routine clinical samples and rectal screening swabs from patients according to the IPC protocol (no informed consent was requested for rectal sampling), cultivated them on blood agar (Becton Dickinson, Heidelberg, Germany) and selective ChromID VRE Agar (Biomerieux, Marcy-l’Étoile, France) and incubated them for 48 h at 37 °C under aerobic conditions. Suspect colonies were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF, Bruker, Bremen, Germany). Antibiotic susceptibility testing was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST version 6.0, 2016) protocol [10 ]. Enterococci were classified as VRE if the minimal inhibitory concentration of vancomycin was above 4 mg/L identified by E-Test (Biomerieux, Marcy-l’Étoile, France) [11 ]. Additional patient data such as diagnosis, duration of hospital stay, outcome, type and time of transplantation were obtained from the medical records of the LKI.
6
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.
7
Bacterial Pneumonia Induction Protocol
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The Streptococci Pneumoniae (ATCC 6305) and Klebsiella Pneumoniae (ATCC 33495) strain were grown respectively on blood agar and McConkey agar (Becton Dickinson, Sparks, MD, USA) for 24 h at 35 0C. Fusobacterium nucleatum (ATCC 25586) strain was grown on Schaedler Broth for 48 h at 35 0C. After it was grown, 1x1010 bacteria (in a 5-mL volume of saline solution) were injected into the right pleural space.
8
Antimicrobial Activities of Methanolic Extracts
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Antimicrobial activities of the methanolic extracts were tested individually against a range of 16 ATCC standard bacteria strains which are clinically important Gram (+) and Gram (-) pathogens. The bacteria tested and antibiotics used as positive controls are listed in Table 3 . Bacterial strains were cultured overnight at 37 °C on Blood Agar (Becton Dickinson, Franklin Lakes, NJ, USA) before testing.
9
Duodenal Fluid Sampling and Microbiome Analysis
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Samples of duodenal luminal fluid were obtained during the EGD, using a sterile double-lumen aspiration catheter (Hobbs Medical, Inc.).31 (link) Aspirates were collected via a sterile inner catheter that was pushed through a sterile bone wax cap only after the second portion of the duodenum was reached, in order to reduce contamination from the mouth, esophagus and stomach. Due to the high viscosity of aspirates, an equal volume of sterile 1x dithiothreitol (DTT) was added to each duodenal aspirate (∼1mL) and the samples were vortexed until fully liquified (∼30 s).31 (link) 100 μl of each sample was then serially diluted with 900 μL sterile 1x PBS and plated on MacConkey agar (Becton Dickinson, Franklin Lakes, NJ, EUA), and on blood agar (Becton Dickinson). Plates were incubated at 37°C for 16-18 hours under aerobic (MacConkey) or anaerobic (blood agar) conditions. Colony forming units (CFU) were then counted electronically using a Scan 500 (Interscience, Paris, France).
The remainder of each sample (the portion not used for microbial culture) was centrifuged at maximum speed (>13000 RPM) for 5 minutes. The supernatant was removed, and 1 mL of sterile Allprotect reagent (QIAGEN, Hilden, Germany) was added to the microbial pellet. The pellets under Allprotect were then stored at −80°C prior to DNA isolation for sequencing.31 (link)
The remainder of each sample (the portion not used for microbial culture) was centrifuged at maximum speed (>13000 RPM) for 5 minutes. The supernatant was removed, and 1 mL of sterile Allprotect reagent (QIAGEN, Hilden, Germany) was added to the microbial pellet. The pellets under Allprotect were then stored at −80°C prior to DNA isolation for sequencing.31 (link)
10
Escherichia coli ATCC 25922 Culture Protocol
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Reference strain Escherichia coli ATCC 25922 (Microbiologics®, Saint Cloud, MN, USA) was used as recommended by the CLSI. Mueller-Hinton broth and agar, Trypticasein-Soy agar, Nutrient agar and Blood agar were acquired from Becton Dickinson (BD, Franklin Lakes, NJ, USA). Soft agar was prepared with 0.6 g agar base (BD, Franklin Lakes, NJ, USA) and 0.6 g NaCl (Promega, Madison, WI, USA).
Saline solution (0.85% NaCl) (J.T. Baker, Phillipsburg, NJ, USA) was used for washing and maintaining cultures. 2% DMSO (J.T. Baker, Phillipsburg, NJ, USA) was used as dissolvent for essential oils. Antibiotics cloramphenicol, cefotaxime, and amikacin were purchased from AMSA (Coyoacan, CDMX, Mexico).
LIVE/DEAD® BacLight™ Bacterial Viability and Counting kit (Cat. L34856, Thermo Fisher, Waltham, MA, USA) was used for sample staining.
McFarland turbidity standard was prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4). All standard preparations were verified in a Coleman spectrophotometer at 600 nm (abs. 0.063) previous to be used.
Saline solution (0.85% NaCl) (J.T. Baker, Phillipsburg, NJ, USA) was used for washing and maintaining cultures. 2% DMSO (J.T. Baker, Phillipsburg, NJ, USA) was used as dissolvent for essential oils. Antibiotics cloramphenicol, cefotaxime, and amikacin were purchased from AMSA (Coyoacan, CDMX, Mexico).
LIVE/DEAD® BacLight™ Bacterial Viability and Counting kit (Cat. L34856, Thermo Fisher, Waltham, MA, USA) was used for sample staining.
McFarland turbidity standard was prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate (BaCl2•2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4). All standard preparations were verified in a Coleman spectrophotometer at 600 nm (abs. 0.063) previous to be used.
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