TITLE: CPE Culture from Companion Animal Rectal Swabs or Feces
AUTHORS: Jackie Dietrich, Stephen Cole

[DESCRIPTION]
Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Our study aimed at optimizing the chromogenic agar method of screening for CPE with and without selective enrichment to isolate CPE from animal feces. The selective enrichment step did not result in any increased recovery of CPE from companion animals with NDM-5 E. coli, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. Therefore, the selective enrichment step is optional, however, this method may not be generalizable to the detection of all types of CPE in fecal specimens from companion animals.

[GUIDELINES]
Samples should be processed within 72 hours of receipt at the laboratory.
Rectal swabs and feces should be refrigerated at 4-8 °C until processed.
All plates should be stored at 4-8°C in a container that protects the agar from light exposure. 
Selective enrichment is optional.

[STEPS]
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
1.       
If starting with feces, dip a cotton tipped applicator into the specimen. The swab does not have to be fully covered with feces, but it should be visibly inoculated. If starting with a rectal swab, proceed to step 2. 
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
2. Using the inoculated swab, streak the first quadrant of the Carba plate.
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
3. Finish streaking the remainder of the quadrants on the plates using a 1-µL inoculation loop.
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
4. Using sterile forceps, add a 10-µg meropenem disc to 5 mL of tryptic soy broth (TSB-MEM).
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
5. Break off the swab or add a pea-sized amount of feces into the TSB-MEM broth. 
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
6. Incubate both the primary plate and inoculated TSB-MEM at 35 ± 2 °C for 18-24 hours.
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
7. After 18-24 hours, observe the primary Carba plate for suspect colonies following manufacturer’s instructions. 
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
7.1. For CHROMID Carba plates, pink-red colonies are presumed to be CPE E. coli while blue-green/grey colonies are presumed to be CPE Klebsiella, Enterobacter, Serratia, or Citrobacter. White, yellow, or tan colonies are presumed to be non-Enterobacterales.
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
8. Using a 10-µL inoculation loop, subculture the inoculated TSB-MEM broth to a secondary Carba plate. 
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
9. Incubate the secondary Carba plate at 35 ± 2 °C for 18-24 hours.
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
10. After 18-24 hours, observe the secondary Carba plate for suspect colonies following manufacturer’s instructions. 
SECTION: CPE Culture from Companion Animal Rectal Swabs or Feces
11. After suspect colonies have been cultured, identification can be performed by subculturing the colony to a blood agar plate then identifying the organism following standard laboratory procedures. Confirmation of carbapenemase production can be performed by modified carbapenem inactivation method (mCIM), EDTA-modified carbapenem inactivation method (eCIM), CarbaNP, NG-Test CARBA 5, CARBA-R PCR, or a lab-developed PCR.