Effect of selected disinfectants on the persistence and movement of a genetically engineered Pseudomonas sp in soil

The effects of disinfectants on survival and movement of Pseudomonas UG14Lr were studied in soil microcosms. Twenty ml bacterial suspension (1.5 x 10(7) CFU/ml) were added to the surface of l-kg soil in microcosms, simulating an accidental release, followed by treatment with 20 ml of 5.0% (w/v) calcium hypochlorite, 75% (v/v) ethanol or 5.0% (v/v) formaldehyde at either 2, 24 or 48 h after inoculation. Microcosms were sampled by removing a soil core which was analyzed at 3 depths for the presence of Pseudomonas sp. UG14Lr as well as total viable and non-viable cells using Lilve/Dead BacLight (Molecular Probes Inc. Oregon, USA), a commercially available fluorescent stain. UG14Lr cells remained concentrated in the upper 3 cm layer of the soil microcosms while decreasing in numbers toward the lower soil layer. However, upon addition of ethanol or formaldehyde, the introduced strain moved toward the middle and lower soil layers. This trend was even more pronounced when disinfectants were added 2 h after inoculation. Pseudomonas sp. UG14Lr cells left in soil for 24 or 48 h before treatment with ethanol or formaldehyde were killed, with the exception of a small number present in the lower soil layer of ethanol-treated microcosms. Calcium hypochlorite did not affect Pseudomonas sp. UG14Lr cell numbers but resulted in a greater concentration of the introduced bacteria in the middle layer of soil microcosms. There were more non-viable cells present at the surfaces of disinfectant-treated soil microcosms compared to the middle and lower layers. Viable cell numbers were slightly reduced at microcosm surfaces upon addition of disinfectants. In ethanol-treated microcosms, viable cell numbers returned to those similar to untreated controls within 2 weeks. These data demonstrated that vertical movement of bacteria may be enhanced upon addition of antimicrobial agents. Additionally, the rime of chemical treatment may be an important consideration in the event of an accidental spill of genetically engineered microorganisms.