Molecular survey of the occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and amoeba hosts in two chloraminated drinking water distribution systems

Abstract

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.

Fig 1

Average copy numbers of Legionella spp., Mycobacterium spp., H. vermiformis, and the 16S rRNA gene in first-draw and postflushing samples. Error bars represent the standard deviations of 29 log-transformed qPCR measurements [log(x + 1)] of target organisms in all first-draw and postflushing samples collected from BCV. ** and ***, significant differences according to paired Wilcoxon rank sum testing at the P < 0.01 and P < 0.001 levels, respectively.

Fig 2

Multidimensional scaling analysis of bacterial community composition (T-RFLP profiles) for postflushing samples from BCV and PCF. Green round symbols represent samples from BCV. Blue inverted triangle symbols represent samples from PCF. Note: one sample from BCV was excluded from analysis due to an absence of T-RFLP peaks.

Fig 3

Multidimensional scaling analysis of bacterial community composition (T-RFLP profiles) for first-draw and postflushing samples from PCF. (A) Each symbol pair represents paired first-draw and postflushing samples from the same location (n = 15). The green circles represent a similarity level of 50% as determined by cluster analysis. (B) Green symbols represent first-draw samples and blue symbols postflushing samples.