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

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). Therefore, to avoid the accidental release of ARGs into environmental water, methods for disinfection of liquid laboratory waste must be effective in destroying nucleic acids. In support of this recommendation, the origin of replication of Enterobacteriaceae plasmids has been detected in strains of non-Enterobacteriaceae bacteria isolated from wastewater from laboratories and research institutes, 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 acid, 6.3% for a commercial P3 disinfectant, 25 min for steam sterilization at 121 °C, and 49 min for disinfection by UVC. A 20-min 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 NaClO-treated cells was 60%, while it decreased under 10% using the commercial disinfectant P3 diluted at 5%. As the degradation was incomplete in both cases, we recommend avoiding discharge of disinfected liquid waste to wastewater (even after chemical neutralization) without additional plasmid destruction treatment, to prevent horizontal transfer of laboratory ARGs to environmental bacteria.

SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.

Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle.

In the present study we developed an enzymatic approach (through the use of collagenase and dispase) to isolate bovine intestinal epithelial cells. Using this method, freshly isolated jejunocytes could be distinguished from simultaneously isolated colonocytes, as the jejunocytes specifically exhibited the small intestinal peptidase gene transcript, as well as an active alkaline phosphatase. The transformation of both types of cell suspension was performed by retroviral infection, using reproduction-defective viruses bearing the gene coding for the large T antigen of the leukaemia simian virus (SV40). The success of the transfection was demonstrated by (1) a significant increase in cell passage numbers (52-53 vs. 7 passages for non-transfected cells), (2) the detection of both the large T transcript and the large T antigen in transformed cells. Possible contamination and progressive substitution of bovine primocultures by non-bovine lineages available in the laboratory was excluded, as the transformed cells presented a bovine typical karyotype. Most transfected cells kept an epithelial morphology after transformation. They also maintained the expression of FABP and enterocyte specific enzymes (brush-border associated maltase and IAP). However, levels of specific activity of these enzymes were low, suggesting that cell differentiation is not completely achieved under the applied culture conditions.

Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures.

BACKGROUND: Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. RESULTS: Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein) as well as an epithelial cytoskeleton component (cytokeratin 18). However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker). Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1-2 mM) or using a glucose-deprived culture medium. CONCLUSION: The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.