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

Abstract

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

Keywords: CPE detection; Isolation; carbapenemase; selective media; sensitivity; specificity.

Figure 1

The whole procedure of method A and method B to compare both and to validate the agar plates. BPW = Buffered Peptone Water; McC + CTX + MEM = MacConkey Agar supplemented with 1 mg/L cefotaxim and 0.125 mg/L meropenem; McC + MEM = MacConkey Agar supplemented with 0.125 mg/L meropenem; LB + CTX = lysogeny broth supplemented with 1 mg/L cefotaxim; LB+MEM = lysogeny broth supplemented with 0.125 mg/L meropenem.

Figure 2

Spreading the sample on the plate. The first spread (blue, first third of the plate) was performed by using a 10 µL-loop. A second spread (green, second third of the plate) was performed by another 10 µL-loop, which was turned the last third of the spread (yellow).

Figure 3

The decreasing number of CPE (E. coli and Salmonella each in average) in pig faeces with and without some additives over ten days.

Figure 4

The final step-by-step guidance of the modified method (method B). BPW = Buffered Peptone Water; McC + CTX + MEM = MacConkey Agar supplemented with 1 mg/L cefotaxim and 0.125 mg/L meropenem; McC + MEM = MacConkey Agar supplemented with 0.125 mg/L meropenem; LB + CTX = lysogeny broth supplemented with 1 mg/L cefotaxim; LB + MEM = lysogeny broth supplemented with 0.125 mg/L meropenem.