After mcr-1 was discovered in 2015, several retrospective studies showed that mcr-1 is widely present in E. coli isolates from livestock, the environment, food, and humans [2, 18–20]. However, the prevalence of E. coli possessing mcr-1 varies widely in foods of animal origin, depending on the sample type and location and time of sampling. For instance, a study showed that the prevalence of mcr-1-harboring E. coli in chicken meat samples collected from the Netherlands between 2009 and 2014 was 1.5% [20], whereas another study on chicken meat samples collected from the same country in 2015 showed a prevalence of 24.8% [21]. Similarly, other studies have shown that the detection rate of E. coli possessing mcr-1 from chicken meat samples collected from Germany [19], China [2], Japan [22], Brazil [23], and Nepal [24] ranged from 1.4–19.5%. More recently, a study in Tunisia showed that the rate of E. coli possessing mcr-1 from a chicken meat sample was relatively high, at 38.3% [25]. However, these detection rates were lower than those found in the present study (66.7%). Likewise, highly variable results have been reported for pork meat samples. The detection rates of E. coli possessing mcr-1 in pork meat samples collected from China [2] and Japan [14] were 19.0% and 3.1%, respectively, while in Germany [19] and the Czech Republic [26], no pork meat sample showed contamination with mcr-1-harboring E. coli.
The detection rate of CR-E. coli from pork and chicken meat samples in this study was higher than that in previous studies; however, the rate is consistent with that in our previous studies from the same area, which showed a high detection rate of mcr-harboring E. coli in fecal samples from healthy humans and livestock (human: 69.39%; chicken: 97.2%; pig: 94.4%) [8, 10]. The magnitude of such variations in the detection rate might largely be because of the sampling conditions mentioned above; moreover, the primary culture medium used for CR bacterial growth seems to affect the results.
As most studies till now have focused merely on the detection rate of mcr-positive CR-E. coli in foods, the quantitative level remains unclear. As a higher density of bacteria in food indicates higher risk in terms of food safety, the quantitative level of CR-E. coli in food is an important indicator of food safety.
Our findings reveal high mean levels of contaminated CR-E. coli in chicken and pork samples. To our knowledge, this is the first study on the quantitative analysis of the contamination of meat samples with CR-E. coli possessing mcr-1.
As E. coli colonies were distinguished from other colonies based on only the color, we recognized these as E. coli-like bacteria in quantitative experiments. However, the representative colonies grown on COL-APSE were isolated from all samples and identified as CR-E. coli by using a standard biochemical identification method, susceptibility tests to colistin, and presence of mcr genes by PCR for the assessment of the contamination rate. The results showed that all these isolates were CR-E. coli possessing mcr-1. Therefore, the results of the quantitative experiments were highly likely to indicate the level of CR-E. coli in food samples, although not all colonies were identified in the experiments.
Both ECC and COL-APSE can be used for quantifying the contamination levels in food samples. However, in some meat samples, the level of contamination with CR-E. coli-like bacteria detected on COL-APSE was even higher than with E. coli-like bacteria detected on ECC. This discrepancy might be caused by the presence of growth factor additives in COL-APSE, which is not disclosed by the manufacturer. Although it is not possible absolutely determine the proportion of CR-E. coli in the contaminated E. coli, with some exceptions, samples with a high number of contaminated E. coli might also have a high number of CR-E. coli in food samples.
Generally, retail meats in local Vietnamese markets are processed as follows: shop owners in rural areas buy pigs and chickens, slaughter the animals themselves, and sell the meat before the day ends. The meat may get contaminated by unhygienic slaughtering processes, the shopkeeper’s hands, and flies or dirt present in the air during sale. Furthermore, because the meat is usually sold in hot and humid environments (average temperature during Vietnamese summers, 26–33 °C; average humidity, > 80%) without hygienic storage facilities, the bacteria can grow easily after contaminating the meat. Considering all these factors, clone population analysis of contaminated CR-E. coli in food is important for understanding the actual situation. The results of PFGE analysis of CR-E. coli isolates revealed that six randomly selected isolates from the same chicken sample belonged to four phylogenetically different clones, implying that these strains can contaminate foods from different sources during various stages of food processing, such as slaughtering, preservation, or sale. In contrast, four of the six E. coli isolates were classified into two phylogenetic groups, indicating that the bacteria may have multiplied after contaminating the food.
Thus, the study showed that meat in local markets was frequently contaminated with certain levels of CR-E. coli. Such highly contaminated meats may contribute to the dissemination of CR-bacteria in the community.