Complementarity of Selective Culture and qPCR for Colistin Resistance Screening in Fresh and Frozen Pig Cecum Samples

Retrospective studies involving the screening of frozen stored collections of samples are commonplace when a new threat emerges, but it has been demonstrated that the freeze-thaw process can affect bacterial viability. The study of colistin-resistant bacteria in human and animal samples is an example of this issue. In this study, we compared culture-based and PCR-based methods for analyzing relative occurrence and diversity of colistin-resistant bacteria in caecal samples to determine the most appropriate method for frozen samples. Thus, 272 samples from the caecal contents of healthy pigs were tested before and after a 6-month freezing period. A selective medium was used when traditional isolation of colistin-resistant bacteria was tested, while a real-time SYBR(®) Green I PCR assay was applied for mcr-1 quantification. The number of samples with colistin-resistant isolates was higher in fresh samples (247/272) than in frozen ones (67/272) and showed a higher diversity of colistin-resistant genera. PCR identification of mcr colistin resistance genes evidenced that mcr-1 was the most prevalent mcr gene and mcr-2 was detected for the first time in pigs from Spanish animal production. The number of samples with mcr-1-carrying bacteria after a freezing period decreased, while real-time quantitation of the mcr-1 gene showed similar values in frozen and fresh samples. Therefore, when frozen cecal samples need to be analyzed, molecular detection of DNA could be the best option to provide a highly representative frame of the initial population present in the sample, and culture-based methods might be a useful complement to study colistin resistance levels.