tet 유전자의 정량화에 의한 어류 장내 미생물총의 Tc 내성 수준 분석

Abstract

This study investigated the proportion of antibiotic-resistant bacteria using quantitative analysis of tetracycline-resistant genes (tet genes) in fish intestines. The proportion of Tc‑resistant bacteria in marine fishes in Korea (20-50%) appeared to be much lower than that of imported freshwater ornamental fishes (70-95%). Additionally, the proportion of Tc‑resistant bacteria from domestic freshwater ornamental fishes (40-60%) was also much lower than that of ornamental fishes from other Asian countries. An analysis of the type of tet genes present in fish microflora found that tet(A) and tet(B) were dominant in Tc-resistant bacteria from ornamental and marine fishes, respectively. ？To determine whether the tet genes were located on plasmids or chromosomes, we performed transformation and conjugation experiments to test the mobility of the tet genes. Transformants and transconjugants containing tet(A) showed tetracycline resistance. The importance of indicator tet gene mobility to other bacteria lies in the possibility that the indicator tet gene may also be located in mobile plasmids and can be transferred to other bacteria. The copy number of tet genes in plasmids was higher than that in chromosomes. ？We report here the development, validation, and use of real-time PCR (qPCR) for both the detection and quantification of various efflux tet genes: tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G), which are common in the microflora of the aquatic environment due to the high use of tetracycline. The copy number of each indicator tet gene was determined using qPCR, with tet(A) used for freshwater ornamental fishes and tet(B) for domestic marine fishes. We also examined the relationship between the frequency of Tc-resistant colonies on coed ntional agar plates and abundances, analyzed using qPCR. These two assays for tet gene load in Tc-resistant bacteria were significantly correlated and evidenceshwaterotential of qPCR for determining the load of Tc-resistant and abundavolving tet genes in aquatic microflora. This study demonstrated that qPCR is not only a rapid diagnosis method for detecting the diverse tet genes in the ecosystem, but also an accurate and sensitive method for comparing the proportion of Tc-resistant bacteria in normal microflora.