TITLE: IDEXX as a Quantitative Detection Method for Antibiotic Resistant (ABR) E. coli
AUTHORS: Gracie Hornsby

[DESCRIPTION]
This SOP describes the validated modification to the standard IDEXX defined substrate assay for detecting both E. coli  and Cefotaxime resistant E. coli in environmental matrices. This method was validated by Hornsby et al., 2022 and shown to be comparable to traditional methods for enumerating antibiotic resistant E. coli in environmental (water, soil, fecal) samples.

[STEPS]
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
1. Follow standard protocol for starting and preparing the laminar flow hood (BSL-2)
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
2. Sterilize bench with 10% bleach solution then 70% ethanol solution
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
3. Pick one colony from the plated standard strain with a disposable loop
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
4. Spin the loop in a 50mL conical tube filled with 10mL of LB broth to wash the colony from the loop into the broth. Repeat with additional tubes if you wish to store additional standards. Dispose of loop in biohazard trash.
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
5. Store the tubes in a shaking incubator (vigorous; ~300rpm) at 37C overnight (12-16 hours)
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
6. After 12-16 hours, store at -80C in a labeled cryobox if you do not wish to use it immediately
SECTION: GROWING AND ESTIMATING E. COLI CONCENTRATIONS FROM A STANDARD STOCK
7. All dilution containers and left-over solution should be placed in a biohazard waste bag then autoclaved on a kill-cycle (at least 121C and 15psi for at least 30 minutes) and disposed of.
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
8. Weigh 100mg (0.1 gram) of Cefotaxime powder into a metal weigh boat (or foil piece) using a sterile scoop
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
9. Add the 100mg of Cefotaxime powder to 20mL of DI water in a sterile 50mL conical tube.
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
10. Mix well (swirling or gentle vortexing) and continue agitating the container until the antibiotic in completely dissolved
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
11. This gives a stock concentration of 5mg/mL.
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
12. Draw up a small amount of air into the syringe, then draw up the entire volume of antibiotic solution
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
13. Attach the filter to the syringe and slowly extrude the solution through the filter into another sterile 50mL conical tube below. Also extrude all the air out of the syringe to clear the filter of all solution.
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
14. Cap the sterilized solution and dispose of the first container, syringe, and filter in a biohazard waste bag.
SECTION: FILTER STERILIZATION OF AN ANTIBIOTIC
15. Label with date, antibiotic name, and initials then store the stock at 4C for up to one month.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
16. CLEARLY mark 120mL vessels and IDEXX trays with organism type (if adding control strains), number if there are duplicates or triplicates (1, 2, 3), antibiotic addition name (CEF added, DI added, or no add), and estimated spiked concentration (10^3, 1-^2, 10^1). Sample label: ESBL EC, CEF added_1, 10^2, processor initials.

SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
17. Follow the standard SOP for environmental samples using IDEXX to prepare samples in 120mL vessels.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
18. If spiking some samples with control strain, follow steps 19-22. Otherwise, skip to step 23.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
19. For samples receiving a control strain spike, 99mL of sample volume should be pipetted into a 120mL vessel rather than the standard 100mL volume.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
20. When ready to use the standard to spike a sample, thaw at room temperature and perform a serial dilution so the spiked sample will be within the IDEXX detectable range (1-2,420 MPN).
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
21. Assuming the standard stocks rest at a concentration of 10^10 organisms after overnight incubation, dilute to obtain an adequate volume of 10^3 stock. Use 5 microfuge tubes filled with 900µL of DI water to create 10^9, 10^8, 10^7, 10^6, and 10^5 dilutions then use a 5mL screw top container to create an adequate volume of 10^4 dilution. Next, use a 50mL conical tube to create the final 10^3 dilution (for each organism) which will be used to spike the samples. 
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
22. Pipette 1mL of 10^3 dilution into 99mL of sample volume to create the desired 10^1 spiked sample solution (total volume of 100mL). Change pipette tip between every sample.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
23. Create lab blanks for each person processing samples by pipetting 100mL of DI water into a labeled 120mL vessel.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
24. Pipette 80µL of Cefotaxime solution (from the 5mg/mL stock solution) into samples which are supposed to receive antibiotic treatment, and recap vessel. Invert a few times to promote mixing. Change pipette tip between each sample. If you wish to obtain the proportion of total E. coli which are antibiotic resistant, two aliquots should be processed from each sample. One aliquot receiving an antibiotic treatment and one aliquot receiving no addition at this step. Lab blanks receive no addition.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
25. Add the Colilert 18 medium to every vessel regardless of treatment, including lab blanks. Continue to agitate intermittently until all medium is completely dissolved. Turn on IDEXX sealer now to ensure it is fully heated.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
26. Pour contents of 120mL vessels into IDEXX trays with normal measures to decrease bubbles and prevent contamination. Tap tray on bench once the sample is poured in to send bubbles to the surface, do not pour sample directly on pull table to prevent cross-contamination.
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
27. Send each tray through the IDEXX sealer on the provided rubber trays with wells facing downward into the rubber tray (paper side facing up). 
SECTION: ENUMERATING ABR E. COLI WITH IDEXX TRAYS
28. Stack the sealed trays with the wells facing down in an incubator for 18-22 hours at 35 degrees C (do not set incubator to shake). Mark date and time in and date and time out on the top tray.
SECTION: COUNTING IDEXX TRAYS
29. Be careful to record the counts in the correct column and row on the data sheet, paying careful attention to whether the tray did or did not receive the antibiotic.
SECTION: COUNTING IDEXX TRAYS
30. Count and record the number of large and small wells which appear yellow under normal laboratory light (does not require UV light chamber).
SECTION: COUNTING IDEXX TRAYS
31. Place in the UV light chamber and count and record the number of large and small fluorescent wells. If some volume of sample overflowed into the large well at the top of the tray, include this in your count as a “large well.” This makes for 48 total small wells and 49 total large wells.
SECTION: COUNTING IDEXX TRAYS
32. Trays with resistant bacteria cannot be disposed of in the normal trash, they must be put in the biohazard trash for autoclaving. Be sure to wipe down counters (bleach and ethanol), turn off the black light, and switch off the used incubator.
SECTION: COUNTING IDEXX TRAYS
33. IDEXX trays should be disposed of by autoclaving on a liquid cycle (minimum of 121C and 15psi for 60 minutes; double-bag all trays and loosely tie both bags). Make sure bags are open enough for steam to penetrate but not so wide that edges could touch the walls of the autoclave.