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Rapid and Practical Screening Method for the Detection of Colistin-Resistant Bacteria in Food

Rapid and Practical Screening Method for the Detection of Colistin-Resistant Bacteria in Food. Biomed J Sci & Res We developed a rapid and practical screening method for simultaneously detecting both Escherichia coli and bacteria harboring the mcr-1 colistin resistance gene using high-speed real-time polymerase chain reaction with specific TaqMan probes. The entire procedure, from sample processing to the final result, was performed within 1 h. The practical utility of this method was verified by analyzing 27 retail meat samples for the presence of colistin-resistant bacteria. The results indicated the potential of this method for the convenient and rapid screening of food items to detect contamination with mcr-1 –positive bacteria, which can be especially useful for on-site testing in developing countries.


Introduction
Colistin is recognized as one of the few remaining available antibiotics for the treatment of intractable infections caused by multidrug-resistant Gram-negative bacteria [1]. Recent studies have shown that bacteria carrying the mcr gene, which confers colistin resistance to most members of the Enterobacteriaceae, are widely disseminated, particularly in Asia [2,3]. Since colistin is widely used in animal husbandry [4], the spread of colistin-resistant (CR) bacteria in communities via livestock food is a potential risk factor. Moreover, CR bacteria are often found in animals and animalfood [5][6][7]; thus, monitoring CR bacteria in animal-food is essential.
However, the conventional culture method [8] for detecting CR bacteria in food is laborious and time-consuming. Rapid detection of colistin resistance genes at the research level is now possible using the SYBR green method [9], but its widespread practicality is limited due to the need for complex steps and equipment involved in DNA extraction from samples and determination of result specificity. To overcome this limitation, we here report a simple, rapid, and practical detection method of Escherichia coli harboring mcr-1, as a representative CR bacterium, using a highspeed real-time polymerase chain reaction (PCR) kit. We further verified the utility of this method for detecting CR bacteria in retail meat samples. Although a real-time PCR assay for the detection of mcr genes from bacterial isolates has already been established, this newly proposed detection method holds practical relevance for widespread use, as the entire procedure, from food sample processing to the final result, can be completed within only 1 h.  coli-like colonies were distinguished based on colony color (dark pink to reddish) after cultivation [8,10]. A representative colony was isolated by sub-culturing on MacConkey agar, and bacterial identification was performed. The colistin minimum inhibitory concentration (MIC) was estimated, and colistin resistance genes (mcr-1 to -5) were detected by multiplex PCR as described previously [6,11].

Materials and Methods
In parallel, DNA was extracted from 1 mL of the homogenate using the Kaneka Easy DNA Extraction Kit version 2 (Kaneka, Tokyo, Japan). The presence of E. coli and the colistin resistance gene mcr-1 in the DNA extracts was determined by real-time PCR using a mobile PCR device, PicoGene PCR1100 (Nippon Sheet Glass, Tokyo, Japan). PCR primers and TaqMan probes for realtime PCR detection of E. coli 16S rRNA and mcr-1 were prepared as described previously (Table 1) [12]. Details regarding the real-

Results and Discussion
The detection sensitivity of the method was assessed using pork meat samples spiked with an mcr-1-positive E. coli strain culture. The lower limit of mcr-1-E. coli detection for the entire method, from DNA extraction to detection by real-time PCR, was 7 × 10 2 CFU/g; however, a minimum of 7 × 10 3 CFU/g was required for quantification using a linear correlation. In the validation study using retail meat samples, CR E. coli-like bacteria were detected using the culture-based method in eight out of ten chicken and in three out of seven pork samples purchased in Vietnam (Table 4).
The semi-quantitative levels of CR bacteria in these samples were in the range 10 3 -10 8 CFU/g ( Table 4). All representative CR E. coli   The detected quantitative mcr-1 levels were higher than the CR E.
coli-like bacterial levels determined via the culture-based method because the real-time PCR method detects all mcr-1 regardless of bacterial species. The quantitative linear range detected via realtime PCR was between 10 3 and 10 6 CFU/g. Although the detected signal was below the quantitative linear range limit in some samples, they were still considered to have positive results via realtime PCR. The approach described in this study provides limited information regarding the degree of contamination; nevertheless, the developed method is reliable and practical owing to a short processing time, enabling the rapid screening of contaminating bacteria with mcr-1 in food.

Conclusion
A new rapid and practical screening method was developed for detecting CR E. coli in food samples. The developed method is advantageous because it is easy to perform, has a short processing time, and provides reliable results that are consistent with those obtained by traditional methods.

Ethics Approval and Informed Consent
Not applicable.