Bacterial Growth State Distinguished by Single-Cell Protein Profiling: Does Chlorination Kill Coliforms in Municipal Effluent?

Municipal effluent is the largest reservoir of human enteric bacteria. Its public health significance, however, depends upon the physiological status of the wastewater bacterial community. A novel immunofluorescence assay was developed and used to examine the bacterial growth state during wastewater disinfection. Quantitative levels of three highly conserved cytosolic proteins (DnaK, Dps, and Fis) were determined by using enterobacterium-specific antibody fluorochrome-coupled probes. Enterobacterial Fis homologs were abundant in growing cells and nearly undetectable in stationary-phase cells. In contrast, enterobacterial Dps homologs were abundant in stationary-phase cells but virtually undetectable in growing cells. The range of variation in the abundance of both proteins was at least 100-fold as determined by Western blotting and immunofluorescence analysis. Enterobacterial DnaK homologs were nearly invariant with growth state, enabling their use as permeabilization controls. The cellular growth states of individual enterobacteria in wastewater samples were determined by measurement of Fis, Dps, and DnaK abundance (protein profiling). Intermediate levels of Fis and Dps were evident and occurred in response to physiological transitions. The results indicate that chlorination failed to kill coliforms but rather elicited nutrient starvation and a reversible nonculturable state. These studies suggest that the current standard procedures for wastewater analysis which rely on detection of culturable cells likely underestimate fecal coliform content.