Pathogens in water systems pose potential health risks. Several countries provide guidelines and risk management strategies for clean water systems. Regarding legionellae, culture-based methods are still the gold standard, whereas molecular methods such as quantitative real-time PCR (qPCR) are controversially discussed among experts as an alternative. It remains questionable as to whether monitoring by qPCR contributes to sustainable water hygiene and effective health prevention. Drinking water samples from 101 buildings were culture-based analyzed to determine the legionellae concentration, along with qPCR tests. The negative predictive values for Legionella spp. and L. pneumophila qPCR regarding the cultivation method were 100% and 98%, respectively. As Legionella spp. DNA is ubiquitously detected, the positive predictive value was low. L. pneumophila DNA was in 18% of the drinking water samples detected by qPCR, among which only 7% was quantifiable. Neither gold-standard methods of cultivation nor qPCR methods alone are suitable to monitor the risk to health by legionellae in water environments adequately. To overcome methodical difficulties, the benefits of a strategic integration of qPCR alongside cultivation methods should be applied to develop a comprehensive protocol for the stepwise analysis of water samples, which can be implemented in international regulatory frameworks in the future.

Legionella species are ubiquitous bacteria found worldwide in water, moist environments, soils, and compost. Infection occurs through the inhalation of aerosols, leading to either Pontiac fever or Legionnaires’ disease (LD). Current routine diagnostics typically combine culture-based isolation with Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for species identification and the Latex Agglutination Test (LAT) for serotyping. However, this workflow is fragmented: MALDI-TOF MS lacks serogroup-specific resolution, while LAT relies on subjective visual interpretation. Therefore, this study evaluated Fourier-transform infrared spectroscopy (FT-IR) as a rapid, high-resolution typing method for Legionella isolates to assess its potential as a single-step diagnostic tool. A total of 200 clinical and environmental Legionella isolates were analyzed using FT-IR, including L. pneumophila serogroups (SG) 1–15 and various non-pneumophila species. Spectral data were analyzed using Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). While MALDI-TOF MS provided accurate species identification, FT-IR spectroscopy demonstrated superior typing capabilities by successfully distinguishing L. pneumophila SG 1 distinct from the SG 2–15 complex and allowing for clear discrimination of most non-pneumophila species. Additionally, FT-IR resolved isolates that showed ambiguous or non-reactive results in LAT. These findings demonstrate that FT-IR overcomes the serotyping limitations of MALDI-TOF MS and offers a more objective, cost-efficient extension to the current multi-step routine, potentially closing the diagnostic gap between simple species identification and deep strain characterization.