Two-stage surveillance of Legionella in environmental water: Optimized multiplex TaqMan qPCR for pathogen detection followed by antimicrobial resistance gene identification

Objectives: Legionellosis, primarily caused by Legionella pneumophila serogroup 1, remains a public health concern in Thailand, particularly in tourism-driven regions. Conventional culture-based surveillance is hindered by limited sensitivity and prolonged turnaround times. This study aimed to develop and validate an optimized multiplex TaqMan real-time PCR assay for simultaneous detection of Legionella spp., Legionella pneumophila , and Legionella pneumophila serogroup 1 in environmental water, while also investigating antimicrobial resistance genes. Methods: A modified primer–probe set targeting the 23S–5S rRNA , mip , and wzm genes was combined with optimized DNA extraction and PCR conditions. Analytical sensitivity was assessed using colony suspensions and spiked water, and specificity was tested against 24 bacterial strains. The assay was validated on 202 environmental water samples collected from four provinces in southern Thailand. Samples positive by real-time PCR were further screened for antimicrobial resistance genes ( lpeAB and tet56 ) using conventional PCR. Results: The optimized assay achieved a detection limit of 1 CFU/mL in suspensions and 1 CFU/L in filtered water, with 100% specificity. Among 202 field samples, PCR detected Legionella in 48.51%, significantly higher than culture (24.26%; p < 0.001). Detection was particularly enhanced in cold-water systems from premise plumbing (41.61% versus 9.49%; p < 0.001). Both methods identified Legionella pneumophila serogroup 1 in 14.29% of samples. Spatial mapping identified Patong subdistrict, Phuket, as a contamination hotspot. Antimicrobial resistance surveillance showed lpeAB in 6.1% of samples and tet56 in one Legionella pneumophila isolate, representing the first report of antimicrobial resistance in environmental Legionella from Thailand. Conclusions: This optimized multiplex real-time PCR assay offers a rapid, sensitive, specific, and cost-effective alternative to culture for Legionella surveillance. The detection of antimicrobial resistance genes highlights potential emerging resistance risks, underscoring the need for integrated molecular and resistance monitoring. Implementation of this approach can strengthen outbreak preparedness and improve water safety management in high-risk settings.