Microbial Diversity in Vehicle Windshield Washer Reservoirs: Findings from Legionella Screening

Legionnaires’ disease remains a relevant public health concern, with transmission linked to droplets from diverse aquatic environments, and its burden across Europe, including in Portugal, has been trending up. Vehicle windshield washer reservoirs have been proposed as potential, yet underexplored, habitats for Legionella spp. In this study, we investigated 62 windshield washer fluid samples collected in central Portugal. Cultivation on selective BCYE agar supplemented with GVPC and subsequent molecular identification revealed no evidence of Legionella spp. However, 23 morphologically distinct bacterial isolates were recovered, and sequencing confirmed diverse taxa from the genera Brevundimonas, Sphingomonas, Ralstonia, and Xanthobacter. These findings indicate that washer reservoirs can sustain microbial communities characterized by environmental resilience and biofilm-forming potential, traits that overlap with ecological niches exploited by Legionella. Although no Legionella was detected, this work represents the first systematic survey of windshield washer reservoirs in Portugal, emphasizing their potential role as overlooked microbial ecosystems and highlighting the importance of continued surveillance. Broader characterization of microbial communities in such artificial aquatic systems may yield insights into microbial interactions that shape pathogen persistence and suppression.