Molecular identification of potential pathogens in water and air of a hospital therapy pool

Indoor warm-water therapy pool workers in a Midwestern regional hospital were diagnosed with non-tuberculosis pulmonary hypersensitive pneumonitis and Mycobacterium avium infections. In response, we conducted a multiseason survey of microorganisms present in this therapy pool water, in biofilms associated with the pool containment walls, and in air immediately above the pool. The survey used culture, microscopy, and culture-independent molecular phylogenetic analyses. Although outfitted with a state-of-theart UV-peroxide disinfection system, the numbers of bacteria in the therapy pool water were relatively high compared with the potable water used to fill the pool. Regardless of the source, direct microscopic counts of microbes were routinely approximate to 1,000 times greater than conventional plate counts. Analysis of clone libraries of small subunit rRNA genes from environmental DNA provided phylogenetic diversity estimates of the microorganisms collected in and above the pool. A survey of >11,300 rRNA genes yielded a total of 628 unique sequences, the most common of which was nearly identical to that of M. avium strains. The high proportion of clones with different Mycobacterium spp. rRNA genes suggested that such organisms comprised a significant fraction of microbes in the pool water (to >30%) and preferentially partition into aerosols (to >80%) relative to other waterborne bacteria present. The results of the study strongly validate aerosol partitioning as a mechanism for disease transfer in these environments. The results also show that culture protocols currently used by public health facilities and agencies are seriously inadequate for the detection and enumeration of potential pathogens.