Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of the Blactx-M Gene In Extended-Spectrum Β-Lactamases (ESBLS)-Producing Enterobacteriaceae Isolated from Sewage of the Philippine General Hospital

Abstract

Extended-spectrum β-lactamase (ESBL)-producing bacteria is considered to be one of the major public health concerns not only in the Philippines but also worldwide. Although common in clinical settings, it has recently been found to increasingly spread among various environments including hospital wastewater. This study designed a Loop-mediated Isothermal Amplification (LAMP) assay for the rapid detection of ESBL-producing bacteria isolated from the sewage of Philippine General Hospital (PGH). First, phenotypic screening of 100 randomly selected Enterobacteriaceae isolates to identify ESBL-producers was conducted. Results showed that 15% of the isolates were phenotypic confirmed as ESBL-producers. Genotyping of the different β-lactamase genes showed that blaCTX-M was the most predominant among the bla genes tested. LAMP primers were then designed for the detection of blaCTX-M using PrimerExplorer v5 with blaCTX-M-1 as the reference genome, while blaCTX-M-2 and blaCTX-M-9 were included in the target group. Primer concentrations, incubation temperature, and reaction time were then optimized, and the resulting sensitivity and specificity were then compared to the conventional Polymerase Chain Reaction (PCR). The designed regular primers have optimal distances, high GC content, and stable at each end. The designed LAMP method has provided similar results to conventional PCR but with only 30 min. of incubation time required. Furthermore, the developed method was comparable with PCR in terms of specificity to blaCTXM gene, as it was able to detect this gene among different members of the Family Enterobacteriaceae. However, the LAMP was 10- to 100-fold more sensitive than the conventional PCR. This study is the first to develop a LAMP assay in the Philippines to detect the presence of ESBL-producing bacteria for environmental surveillance. The study demonstrates the feasibility of the LAMP assay for the rapid detection of ESBL genes in environmental samples.