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Abstract Understanding how Legionella spp. proliferate in multispecies biofilms is essential to develop strategies to control their presence in building plumbing. Here, we analyzed biofilm formation and Legionella spp. colonization on new plumbing material during 8 weeks. Biofilm formation was characterized by an initial increase in intact cell concentrations up to 9.5 × 105 cells/cm2, followed by a steady decrease. We identified Comamonas, Caulobacter, Schlegella, Blastomonas and Methyloversatilis as pioneer genera in the biofilm formation process. Importantly, L. pneumophila was the dominant Legionella spp. and rapidly colonized the biofilms, with culturable cell concentrations peaking at 3.1 × 104 MPN/cm2 after 4 weeks already. Moreover, several Legionella species co-occurred and had distinct dynamics of biofilm colonization. Vermamoeba vermiformis (V. vermiformis) was the dominant protist identified with 18S rRNA gene amplicon sequencing. Together our results highlight that biofilm formation upon introduction of new building plumbing material is a dynamic process where pathogenic Legionella species can be part of the earliest colonizers.

When stay-at-home orders were issued to slow the spread of COVID-19, building occupancy (and water demand) was drastically decreased in many buildings. There was concern that widespread low water demand may cause unprecedented Legionella occurrence and Legionnaires’ disease incidence. In lieu of evidenced-based guidance, many people flushed their water systems as a preventative measure, using highly variable practices. Here, we present field-scale research from a building before, during, and after periods of low occupancy, and controlled stagnation experiments. We document no change, a > 4-log increase, and a > 1.5-log decrease of L. pneumophila during 3- to 7-week periods of low water demand. L. pneumophila increased by > 1-log after precautionary flushing prior to reoccupancy, which was repeated in controlled boiler flushing experiments. These results demonstrate that the impact of low water demand (colloquially called stagnation) is not as straight forward as is generally assumed, and that some flushing practices have potential unintended consequences. In particular, stagnation must be considered in context with other Legionella growth factors like temperature and flow profiles. Boiler flushing practices that dramatically increase the flow rate and rapidly deplete boiler temperature may mobilize Legionella present in biofilms and sediment.

Abstract Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen‐fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH‐N cycle as a novel metabolic pathway that co‐catabolizes plant‐provided arginine and succinate to drive the energy‐demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH‐N cycle including transaminases that interlink the co‐catabolism of arginine and succinate. The CATCH‐N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism—two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH‐N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH‐N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant‐associated organisms with new properties to improve nitrogen fixation.