Heterotrophic bacteria were enumerated from structural surfaces, drain-pan water and the air stream of a well maintained air-handling system with no reported building-related illness. Visually the system appeared clean but large populations of bacteria were found on the fin surface of the supply-side cooling coils (105-106 CFU cm-2), in drain-pan water (105-107 CFU ml-1), and in sump water of the evaporative condenser (105 CFU ml-1). Representative bacterial colony types were recovered from heterotrophic plate counts on R2A and identified to genus level. Pink-pigmented budding rods (PPBR) phenotypically resembling members of the genus Blastobacter dominated the supply surface of the coll fins, the drain-pan water and post-coil air. This data and independent scanning electron microscopy were consistent with the presence of a resident population of predominantly PPBR bacteria in a biofilm on the supply-side cooling coil fins.
The primary colonizing and mature biofilm communities of the cooling coil fins were recovered from the cooling coils of the study system. Primary colonizers were recovered on sterile peroxide-cleaned alloy coupons inserted between coil fins at the base of the supply-side of the cooling coil bank for brief periods of time. Cells adhering to alloy coupons and in situ coil fin alloy were removed by shearing them onto an agar surface where they could grow into colonies. Fifteen bacterial colony types were identified from the primary colonizing and mature biofilm communities. Of these, eight colony types were consistently recovered as primary colonizers of the coil fin alloy (core group) and seven colony types were only recovered infrequently (peripheral group). Four members of the core group were phenotypically identified; Methylobacterium sp. (pink colony type), Alcaligenes paradoxus (green spreader type), Sphingomonas paucimobilis (bright yellow group 2 type) and Sphingobacterium sp. (orange-pink type). The four unidentified members of the core group were all gram-negative rods. The pink colony type was phenotypically identical to the numerically dominant member of the coil fin biofilm isolated in the initial study. It also appeared to be the dominant member of the colonizing community.
Five of the seven members of the peripheral group were phenotypically identified; Methylobacterium spp. (red-pink and pale pink types), Corynebacterium sp. (bright yellow groups type), Bacillus megaterium (Bacillus type 1) and Bacillus cereus (Bacillus type 2). The two unidentified peripheral group members were both gram-positive rods.
Direct counts of bacteria adhering to the coil fin alloy substratum after 1, 5 or 30 minutes exposure to the cooling coil habitat indicated that colonization of the alloy was very rapid with approximately 7x104 cells adhering per square cm of alloy after only 1 minutes exposure. In addition, all colony types in the core group had colonized the coil fin alloy in 1 minute. This rapid colonization of clean coil fin alloy by a wide range of bacterial types was consistent with large numbers of bacteria being shed into the coil fin condensate from mature coil fin biofilm.
The phylogenetic positions of the coil fin colony types were determined by sequencing the 16S rRNA gene of representative strains. Comparison of near-complete sequences revealed that the pink, red-pink and pale pink colony types, collectively termed PPBR, were most closely related to the genus Methylobacterium in the alpha proteobacterial subclass (alpha-2 group), but distinct from previously sequenced representatives of this genus. Comparison of partial sequences (nucleotide positions 144 to 485 of the E. coli gene) of the other coil fin isolate colony types indicated the core group contained three members belonging to the alpha proteobacterial subclass apart from the pink colony type. They were all members of the alpha-4 group; bright yellow groups 1 and 2 (unidentified and Sphingomonas paucimobilis respectively) and the orange colony type (unidentified). The core group also contained two members belonging to the beta proteobacterial subclass; green spreader (Alcaligenes paradoxus) and off-white (unidentified) and two members belonging to the Bacteroides-Cytophaga branch; pale yellow (unidentified) and orange-pink (Sphingobacterium sp.). Apart from the two Methylobacterium -like colony types, the peripheral group contained two members belonging to the Actinomycetes branch; green (unidentified) and bright yellow group 3 (Corynebacterium sp.) and two members belonging to the Clostridium-Bacillus branch; Bacillus type 1 (B. megaterium) and Bacillus type 2 (B. cereus). The seventh peripheral group member, the white mucoid colony type, was unamenable to both phenotypic and phylogenetic identification. The identities of the phenotypically identified colony types were consistent with their phylogenetic placements.
Two hypotheses as to why the pink Methylobacterium -like colony type should apparently dominate the coil fin biofilm were tested. The first hypothesis was that the pink colony type possessed pronounced cell surface hydrophobicity (CSH) which enabled it to colonize the coil fin alloy and dominate the biofilm from colonization to climax community. The second hypothesis was that the pink colony type had pronounced desiccation resistance which allowed it to survive the winter months when the cooling coils were dry for most of the time.
Cells of the pink colony type appeared to have one hydrophobic pole as determined by binding of unconjugated colloidal gold particles. This was consistent with relatively high total population CSH as determined by the microbial adhesion to hydrocarbons (MATH) and hydrophobic interaction chromatography (HIC) assays. However, the pink colony type did not have pronounced CSH relative to other less populous primary colonizer isolates tested, such as the pale yellow and bright yellow group 2 colony type strains. Staining of pink colony type cells (strain BF15) with fluorescently labelled wheat germ agglutinin suggested that the cells have polar holdfasts which contain N -acetyl glucosamine oligomers similar to previously described polar holdfasts in Caulobacter species. The holdfast was noted to anchor PPBR cells in rosettes.
Pink colony type strains had pronounced desiccation resistance relative to all other colony type representives tested with the exception of the red-pink and pale pink Methylobacterium -like colony types. This suggests the PPBR as a group are desiccation resistant possibly due to a thick peptidoglycan layer (10 nm) observed in a thin section of a pink colony type strain (BF15). The desiccation resistance of the PPBR strains is consistent with known characteristics of strains of the genus Methylobacterium which are relatively tolerant to desiccation and representatives of which have been found in dry environments such as road-side dust. Hypotheses concerning the community structure and origin of the coil fin biofilm based on the data presented above are discussed.