Development of an RT-LAMP Assay for Detecting tet(M) in Enterococcus Species: Enhancing AMR Surveillance Within the One Health Sectors

Abstract

The increasing prevalence of antimicrobial-resistant (AMR) bacteria in humans, animals, and the environment underscores the necessity for a rapid, sensitive, and specific method to identify resistance genes. Objectives: This study aims to develop a reliable detection tool for identifying the tetracycline-resistant gene tet(M) in Enterococcus species using a real-time loop-mediated isothermal amplification (RT-LAMP) assay. Real-time visualization through a turbidimeter enabled precise estimation of time-to-positivity for gene detection. Methodology: Six primers were designed using PrimerExplorer v.5, and the assay was optimized across different temperatures and incubation times. Validation was conducted by testing 52 tet(M)-positive clinical enterococci isolates and spiking urine samples from a healthy volunteer and a cow with tet(M)-positive Enterococcus species. Results: The tet(M) gene was detected as early as 33 min, with optimal amplification occurring within 60 min at 60 °C. The assay demonstrated 100% specificity with the established primers. The sigmoidal graphs were corroborated with visual confirmation methods, including a green color change (visible to the naked eye), green fluorescence (under UV light), and a 200 bp PCR product observed via agarose gel electrophoresis. Notably, the tet(M) RT-LAMP assay exhibited a detection limit of 0.001 pg/μL, significantly surpassing conventional PCR, which had a detection limit of 0.1 pg/μL. Conclusions: This rapid, cost-effective, highly sensitive, and specific tet(M) RT-LAMP assay holds significant promise as a surveillance tool for antimicrobial resistance monitoring within a One Health framework, particularly in low-resource countries.

Keywords: One Health; RT-LAMP; antimicrobial-resistant; enterococci; tet(M); tetracycline resistance.

Figure 1

Designation of LAMP primers. The loci of the nucleotide sequences (purple arrows) were used for the loop-mediated isothermal amplification (LAMP) primers. These primers targeted the tetracycline resistance tet(M) gene in enterococci. F3 and B3 were used as the outer primers, while FIB and BIP were chosen as the inner primers. LB and LF (loop primers) were used to amplify the reaction.

Figure 2

Optimization of tet(M) RT-LAMP assay according to temperatures using four techniques for detecting RT-LAMP assay products (A–D). (A) Lines with different colours represented the generation of PCR products at different temperature readings at 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, and 65 °C. (B,C) Colour changes from orange to green and green fluorescence (calcein dye) with UV light were observed in the reaction tubes. (D) The LAMP PCR products of 200 bp were observed on 1.5% gel electrophoresis. M: a 100 bp ladder (Eco Plus, Selangor, Malaysia), PC: positive control (E. faecalis ATCC 29212), NC: negative control (distilled water).

Figure 3

(A) Complete formation of sigmoidal graphs within 45 and 60 min (orange and blue line colours). (B,C) The colour changes from orange to green, and the formation of green fluorescence was observed by the naked eye and under UV light, respectively. (D) A 200 bp of PCR products were observed on 1.5% gel electrophoresis. M: a 100 bp ladder (Eco Plus, Selangor, Malaysia), PC: positive control (E. faecalis ATCC 29212), NC: negative control (distilled water).

Figure 4

(A) Two sigmoidal graphs generated by the amplification of the tet(M) gene of the E. faecalis ATCC 29212 (purple line) and E. faecalis clinical (blue line). (B,C) The color changes from orange to green, and the formation of green fluorescence was observed by the naked eye and under UV light, respectively. (D) Only two 200 bp PCR bands were observed in Lanes 1 and 2. M: a 100 bp ladder (Eco Plus, Selangor, Malaysia), Lane 1: E. faecalis ATCC 29212, Lane 2: E. faecalis clinical isolate), Lane 3: K. pneumoniae ATCC 1705, Lane 4: S. pyogenes ATCC 19615, Lane 5: S. agalactiae NCTC 8017, Lane 6: E. coli ATCC 25922, Lane 7: P. aeruginosa ATCC 27853, Lane 8: K. pneumoniae ATCC 700603, Lane 9: S. aureus ATCC 25923, Lane 10: E. gallinarum clinical isolate, and Lane 11: E. faecalis clinical isolate.

Figure 5

This figure presents a comparative analysis of the sensitivity of RT-LAMP and conventional PCR assays for detecting the tet(M) gene in Enterococcus faecalis ATCC 29212. A 200 bp PCR product was observed on 1.5% gel electrophoresis. Lane M contains a 100 bp ladder (Eco Plus, Selangor, Ma-laysia); Lanes 1–9 represent decreasing DNA concentrations (10² to 10⁻⁶ ng/µL); PC denotes the positive control (E. faecalis ATCC 29212, undiluted), and NC indicates the negative control (distilled water).

Figure 6

The validation performance of tet(M) RT-LAMP assay in spiked human urine. (A) The formation of sigmoidal graphs with different colours was observed from all reaction tubes. (B,C) Visual observations by the colour change from orange to green and green fluorescence through the naked eye and under the UV light, respectively. (D) A 1.5% gel electrophoresis revealed 200 bp PCR bands. M: a 100 bp ladder (Eco Plus, Selangor, Malaysia). Lane M: a 100 bp ladder (Eco Plus, Selangor, Malaysia), Lane 1: E. faecalis, Lane 2: E. faecalis, Lane 3: E. faecalis, Lane 4: E. faecalis, Lane 5: E. faecium, Lane 6: E. faecium, Lane 7: E. faecium, Lane 8: E. gallinarum, Lane 9: E. gallinarum, Lane 10: E. gallinarum, and Lane 11: E. casseliflavus PC: positive control (E. faecalis ATCC 29212), NC: negative control (distilled water).

Figure 7

The validation performance of tet(M) RT-LAMP assay in spiked animal urine. (A) Sigmoidal graphs with different colours were generated from all reaction tubes. (B,C) The presence of colour changes from green to yellow and green fluorescence was observed by the naked eye and under UV light, respectively. (D) PCR products of 200 bp were observed on a 1.5% gel electrophoresis. M: a 100 bp ladder (Eco Plus, Selangor, Malaysia), Lane M: a 100 bp ladder (Eco Plus, Selangor, Malaysia), Lane 1: E. faecalis, Lane 2: E. faecalis, Lane 3: E. faecalis, Lane 4: E. faecalis, Lane 5: E. faecium, Lane 6: E. faecium, Lane 7: E. faecium, Lane 8: E. gallinarum, Lane 9: E. gallinarum, Lane 10: E. gallinarum, and Lane 11: E. casseliflavus PC: positive control (E. faecalis ATCC 29212), NC: negative control (distilled water).