Complementarity of selective culture and qPCR for colistin resistance screening in fresh and frozen pig caecum samples
Investigación publicada en Frontiers in Microbiology
9 de noviembre de 2020
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 analysing both relative occurrence and diversity of colistin-resistant bacteria in caecal samples to determine the most appropriate method for frozen samples. Thus, 272 isolates from the caecal contents of healthy pigs were tested before and after a six-month freezing period. A selective medium was used when traditional isolation of colistin-resistant bacteria was tested, while a real-time SYBR Green 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 also 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 caecal samples need to be analysed, 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
Miguela-Villoldo P., Moreno MA., Hernandez M., Rodriguez-Lazaro D., Gallardo A., Borge C., Quesada A., Dominguez L. y Ugarte-Ruiz M..
 | Servicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM). |
| Departamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM). |
 | Instituto Tecnológico Agrario de Castilla y León (ITACyL). |
 | Universidad de Burgos (UBU). |
 | Facultad de Veterinaria. Universidad de Extremadura (UNEX). |
 | Facultad de Veterinaria. Universidad de Córdoba (UCO). |
| Instituto Universitario de Biotecnología Ganadera y Cinegética (INBIO G+C). Universidad de Extremadura (UNEX). |
