Isolation Procedure for CP E. coli from Caeca Samples under Review towards an Increased Sensitivity.

Area of Science:

• Microbiology
• Food Safety
• Antimicrobial Resistance

Background:

• Carbapenemase-producing Enterobacteriaceae (CPE) are increasingly reported in livestock.
• Existing European Reference Laboratory for Antimicrobial Resistances (EURL-AR) protocols have limitations in detecting CPE with low carbapenem Minimum Inhibitory Concentration (MIC) values.
• Accurate detection of CPE in food-producing animals is crucial for public health and preventing antimicrobial resistance spread.

Purpose of the Study:

• To modify and enhance the EURL-AR protocol for improved recovery of CPE from livestock samples.
• To evaluate the impact of a second selective enrichment step and in-house prepared selective agar on CPE detection sensitivity and specificity.
• To optimize the detection of CPE, particularly those with low-level carbapenem resistance.

Main Methods:

• A modified protocol included a second selective enrichment step using lysogeny broth supplemented with cefotaxime (CTX) and meropenem (MEM) under microaerophilic conditions.
• Real-time PCR was employed to pre-screen enrichments for common carbapenemase genes.
• In-house prepared MacConkey agar with MEM and MEM+CTX was compared against commercial selective agar for CPE isolation.

Main Results:

• The use of in-house prepared selective agar achieved 100% sensitivity and specificity, a significant improvement over commercial agar which yielded ~75% sensitivity.
• The second enrichment step did not substantially influence overall sensitivity and specificity but simplified the isolation process by reducing accompanying microbiota.
• The modified protocol demonstrated enhanced capabilities for isolating CPE, including those with low carbapenem MIC values.

Conclusions:

• Slight modifications to the EURL-AR protocol, particularly the use of in-house selective media, can significantly increase the sensitivity for detecting carbapenemase-producing Enterobacteriaceae (CPE) in livestock.
• The enhanced method aids in the simplified isolation of CPE and the separation of target colonies.
• The optimized protocol is valuable for surveillance and control of CPE in the food production chain.