Frontiers in Microbiology (Nov 2020)

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

  • Pedro Miguela-Villoldo,
  • Pedro Miguela-Villoldo,
  • Miguel A. Moreno,
  • Miguel A. Moreno,
  • Marta Hernández,
  • David Rodríguez-Lázaro,
  • Alejandro Gallardo,
  • Carmen Borge,
  • Alberto Quesada,
  • Alberto Quesada,
  • Lucas Domínguez,
  • Lucas Domínguez,
  • María Ugarte-Ruiz

DOI
https://doi.org/10.3389/fmicb.2020.572712
Journal volume & issue
Vol. 11

Abstract

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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.

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