發(fā)布者：抗性基因網(wǎng) 時(shí)間：2023-06-14 瀏覽量：2014
摘要
? ? ? 任何分子生物學(xué)實(shí)驗(yàn)室的日常工作都包括日常使用微生物，包括用多種表達(dá)至少一種抗生素抗性基因（ARG）的質(zhì)粒轉(zhuǎn)化的大腸桿菌菌株。
為了驗(yàn)證消毒方法對(duì)實(shí)驗(yàn)室液體廢物的有效性，通過(guò)16S核糖體RNA測(cè)序鑒定了從實(shí)驗(yàn)室和研究所排水溝中分離的細(xì)菌，并測(cè)試了實(shí)驗(yàn)室質(zhì)粒中常見(jiàn)的復(fù)制來(lái)源和幾種ARG的存在。令人驚訝的是，在非腸桿菌科細(xì)菌菌株中檢測(cè)到腸桿菌科質(zhì)粒的復(fù)制來(lái)源，這表明實(shí)驗(yàn)室質(zhì)粒已經(jīng)發(fā)生了種間轉(zhuǎn)移。
使用定量聚合酶鏈?zhǔn)椒磻?yīng)，我們測(cè)定了幾種去污方法對(duì)用卡那霉素抗性質(zhì)粒（pET28A?+?或pEGFP-C2）。硫酸的D值估計(jì)值為0.7 M，商用P3消毒劑的D值為6.3%，121°C蒸汽滅菌25分鐘，紫外線滅菌49分鐘。用最終濃度為1-10%的次氯酸鈉處理細(xì)菌培養(yǎng)物20分鐘，發(fā)現(xiàn)在完全破壞細(xì)菌質(zhì)?；驑?biāo)記物（編碼pBR322復(fù)制起源）方面無(wú)效。來(lái)自HClO處理的細(xì)胞的殘余DNA為60%，而使用稀釋為5%的商用消毒劑P3，其減少到10%以下。由于這兩種情況下的降解都不完全，為了防止實(shí)驗(yàn)室ARGs水平轉(zhuǎn)移到環(huán)境細(xì)菌中，如果沒(méi)有額外的質(zhì)粒破壞處理，消毒后的液體廢物不應(yīng)釋放到污水中。
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.
https://www.researchsquare.com/article/rs-2609208/v1