Distribution of Legionella and bacterial community composition among regionally diverse US cooling towers

Abstract

Cooling towers (CTs) are a leading source of outbreaks of Legionnaires' disease (LD), a severe form of pneumonia caused by inhalation of aerosols containing Legionella bacteria. Accordingly, proper maintenance of CTs is vital for the prevention of LD. The aim of this study was to determine the distribution of Legionella in a subset of regionally diverse US CTs and characterize the associated microbial communities. Between July and September of 2016, we obtained aliquots from water samples collected for routine Legionella testing from 196 CTs located in eight of the nine continental US climate regions. After screening for Legionella by PCR, positive samples were cultured and the resulting Legionella isolates were further characterized. Overall, 84% (164) were PCR-positive, including samples from every region studied. Of the PCR-positive samples, Legionella spp were isolated from 47% (78), L. pneumophila was isolated from 32% (53), and L. pneumophila serogroup 1 (Lp1) was isolated from 24% (40). Overall, 144 unique Legionella isolates were identified; 53% (76) of these were Legionella pneumophila. Of the 76 L. pneumophila isolates, 51% (39) were Lp1. Legionella were isolated from CTs in seven of the eight US regions examined. 16S rRNA amplicon sequencing was used to compare the bacterial communities of CT waters with and without detectable Legionella as well as the microbiomes of waters from different climate regions. Interestingly, the microbial communities were homogenous across climate regions. When a subset of seven CTs sampled in April and July were compared, there was no association with changes in corresponding CT microbiomes over time in the samples that became culture-positive for Legionella. Legionella species and Lp1 were detected frequently among the samples examined in this first large-scale study of Legionella in US CTs. Our findings highlight that, under the right conditions, there is the potential for CT-related LD outbreaks to occur throughout the US.

Fig 1. Cooling tower locations and PCR and culture results overview.

The CT samples in this study were from geographic locations across the US (A). The approximate longitude and latitude of the city for each CT location is plotted on the map which was generated with SimpleMappr (www.simplemappr.net). CTs that were positive for Legionella DNA by PCR are represented by red triangles [PCR (+)] and those negative by PCR are shown as blue circles [PCR (-)] (A). PCR (+) refers to a positive result for any of the three discrete probes in the PCR assay (Lspp, Lp, Lp1) (A). Each sample was assayed by PCR (A, B) and PCR-positive samples underwent culture (B). Samples that yielded no isolates were culture-negative [Culture (-)]. PCR-negative samples were not cultured (NC).

Fig 2. Geographic distribution of results.

Samples from eight US climate regions [Central (C), East North Central (ENC), Northeast (NE), Northwest (NW), South [Central] (SC), Southeast (SE), Southwest (SW), and West (W)] were assayed by PCR (A) and culture (B). Specimens that were positive for any of the targets by PCR underwent culture. Isolates were identified to the species (C) and L. pneumophila serogroup (D) level.

Fig 3. Samples with higher diversity of taxa correlate with higher levels of Legionellaceae abundance.

PCR positive samples were analyzed by 16S rRNA amplicon sequencing and the relative Legionellaceae abundance of each was compared to the number of bacterial families (relative abundance >1%) detected and organized by initial sample multiplex PCR result (A). The relative Legionellaceae abundance (B) and number of taxa (C) of samples that were positive or negative for multiplex PCR and culture were analyzed. Bars represent the geometric (B) or arithmetic (C) mean and error bars indicate standard deviation.

Fig 4. Phyla and Proteobacteria family profiles related to PCR and culture results.

The composition of the five most abundant phyla (A, B) and five most abundant Proteobacteria families (C, D) present in cooling tower water samples were compared by overall Legionella PCR result (A, C) and culture result (B, D). Note that only samples that were positive by PCR underwent culture.

Fig 5. Comparison of Pinellas County CT bacterial family compositions between spring and summer 2016.

Multiplex PCR results, culture results, and five highest abundance Proteobacteria family compositions were compared between spring (A) and summer (B) 2016. These samples were PCR-positive for Lspp marker only (Lspp) or all three Lspp, Lp, and Lp1 markers (Lp1). Samples were culture-positive for non-Lp species (Lspp), Lp1 isolates only (Lp1), or were culture-negative [(-)]. In samples where Legionella spp. were detected by PCR, Legionellaceae was not always among the bacterial families with the highest abundance.