Prevention of horizontal transfer of laboratory plasmids to environmental bacteria: comparison of the effectiveness of a few disinfection approaches to degrade DNA.

Abstract

The routine work of any molecular biology laboratory includes the daily use of microorganisms, including strains of E. coli, transformed with a variety of plasmids expressing at least one antibiotic resistance gene (ARG). To verify the effectiveness of disinfection methods on laboratory liquid waste, bacteria isolated from laboratory and research institute drains were identified by 16S ribosomal RNA sequencing and tested for the presence of an origin of replication and several ARGs frequently found in laboratory plasmids. Surprisingly, the origin of replication of Enterobacteriaceae plasmids was detected in strains of non-Enterobacteriaceae bacteria suggesting that interspecific transfer of laboratory plasmids had occurred. Using quantitative Polymerase Chain Reaction, we determined the Decimal reduction value (D-value, expressed as concentration of disinfectant or length of physical treatment) of several decontamination methods for their DNA degradation effect on cultures of E. coli Top10 transformed with a kanamycin resistant plasmid (pET28A + or pEGFP-C2). The estimated D-values were 0,7 M for Sulfuric, 6,3% for a commercial P3 disinfectant, 25 minutes for steam sterilization at 121°C and 49 minutes for disinfection by UVC. A 20-minute treatment of bacteria cultures with a final concentration of 1–10% sodium hypochlorite was found to be ineffective in completely destroying a bacteria plasmid gene marker (coding for the pBR322 origin of replication). Residual DNA from HClO treated cells was 60%, while it decreased under 10% using the commercial disinfectant P3 diluted at 5%. As the degradation was uncomplete in both cases, to prevent the horizontal transfer of laboratory ARGs to environmental bacteria, disinfected liquid waste should not be released in sewage without additional plasmid destruction treatment.