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.
