Abstract
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.
Highlights
Retrospective studies involving the screening of stored frozen collections of isolates or, less frequently, biological samples are commonplace when a new microbiological threat emerges, and their aim is to test prior occurrences and features of the agent
ACRB isolates were detected in 222/272 of fresh samples, (82%) and 60/272 of frozen-thawed samples (22%), whereas NCRB isolates were obtained from 140/272 of fresh samples (51%) and 12/272 of frozen-thawed samples (4%) (p-value < 0.001)
Regarding both ACRB and NCRB groups, we noticed that the freeze-thaw process affected these two groups differently, since NCRB (192 in fresh samples vs. 15 in frozen samples) showed a higher decrease in the number of isolates obtained than ACRB (294 in fresh samples vs. 63 in frozen samples), further analyses are needed to determine the cause
Summary
Retrospective studies involving the screening of stored frozen collections of isolates or, less frequently, biological samples are commonplace when a new microbiological threat emerges, and their aim is to test prior occurrences and features of the agent. The freeze-thaw process could be an important issue for bacterial recovery since it could dramatically affect bacterial survival because of the physical changes in the frozen material, assuming that at least a 74% decrease in cell viability may occur (Phalakornkule et al, 2017). This is of paramount importance when the aim is testing bacterial diversity of frozen samples, as previous studies have demonstrated that the freeze-thaw process can decrease bacterial diversity in the samples (Phalakornkule et al, 2017; Dorsaz et al, 2020). The bacterial composition of stools was not dramatically affected after a freezing process using molecular methods, this has been associated with freezing conditions (Shao et al, 2012)
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