Toxoflavin secreted by Pseudomonas alcaliphila inhibits growth of Legionella pneumophila and its host Vermamoeba vermiformis

Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers contaminated with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. The genus Pseudomonas correlates negatively with the presence of L. pneumophila, but it is not clear which species is responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. Cooling towers free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. In vitro, P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a genes cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extract from P. alcaliphila culture found to contain toxoflavin by LC-ESI-MS. In addition, toxoflavin inhibits growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration is likely not achieved in the bulk water but might have a local inhibitory effect such as in biofilm.

Water Age Effects on the Occurrence and Concentration of Legionella Species in the Distribution System, Premise Plumbing, and the Cooling Towers

In this study, droplet digital PCRTM (ddPCRTM) was used to characterize total Legionella spp. and five specific Legionella species from source (groundwater) to exposure sites (taps and cooling towers). A total of 42–10 L volume water samples were analyzed during this study: 12 from a reservoir (untreated groundwater and treated water storage tanks), 24 from two buildings (influents and taps), and six from cooling towers, all part of the same water system. The approximate water age (time in the system) for all sample locations are as follows: ~4.5, 3.4, 9.2, 20.8, and 23.2 h (h) for the groundwater to the reservoir influent, reservoir influent to the reservoir effluent, reservoir effluent to building Fa (building names are abbreviated to protect the privacy of site location), building ERC and the cooling towers, respectively. Results demonstrated that gene copies of Legionella spp. (23S rRNA) were significantly higher in the cooling towers and ERC building (p < 0.05) relative to the reservoir and building Fa (closest to reservoir). Legionella spp. (23S rRNA) were found in 100% (42/42) of water samples at concentrations ranging from 2.2 to 4.5 Log10 GC/100 mL. More specifically, L. pneumophila was found in 57% (24/42) of the water samples, followed by L. bozemanii 52% (22/42), L. longbeachae 36% (15/42), L. micdadei 23% (10/42) and L. anisa 21% (9/42) with geometric mean concentrations of 1.7, 1.7, 1.4, 1.6 and 1.7 Log10 GC/100 mL, respectively. Based on this study, it is hypothesized that water age in the distribution system and the premise-plumbing system as well as building management plays a major role in the increase of Legionella spp., (23S rRNA) and the diversity of pathogenic species found as seen in the influent, and at the taps in the ERC building—where the building water quality was most comparable to the industrial cooling towers. Other pathogenic Legionella species besides L.pneumophila are also likely amplifying in the system; thus, it is important to consider other disease relevant species in the whole water supply system—to subsequently control the growth of pathogenic Legionella in the built water environment.

Legionella Occurrence beyond Cooling Towers and Premise Plumbing

Legionella is an environmental pathogen that is responsible for respiratory disease and is a common causative agent of water-related outbreaks. Due to their ability to survive in a broad range of environments, transmission of legionellosis is possible from a variety of sources. Unfortunately, a disproportionate amount of research that is devoted to studying the occurrence of Legionella in environmental reservoirs is aimed toward cooling towers and premise plumbing. As confirmed transmission of Legionella has been linked to many other sources, an over-emphasis on the most common sources may be detrimental to increasing understanding of the spread of legionellosis. This review aims to address this issue by cataloguing studies which have examined the occurrence of Legionella in less commonly investigated environments. By summarizing and discussing reports of Legionella in fresh water, ground water, saltwater, and distribution system drinking water, future environmental and public health researchers will have a resource to aid in investigating these pathogens in relevant sources.