ABSTRACTThe 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, andPseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence ofLegionella(≥69.0%) and mycobacteria (100%), lower occurrence ofL. pneumophila(≤20%) andM. avium(≤33.3%), and rare detection ofPseudomonas aeruginosa(≤13.3%) were observed in both systems according to quantitative PCR. Also,Hartmanella vermiformiswas more prevalent thanAcanthamoeba, 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 ofLegionella, 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. vermiformisand total bacteria, mycobacteria andH. vermiformis, andLegionellaandH. 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.