Spread Of Animal Original ESBL-Producing Escherichia Coli And The Promotion Effect And Mechanism Of Bentonite On ESBL Gene Transfer

Extended-spectrum beta-lactamases(ESBLs) are enzymes that can hydrolyze the penicillin, the first-, second-, and third-generation cephalosporin, and aztreonam except cephamycins or carbapenems, but susceptible to inhibitors. The bacteria carrying ESBLs could destroy these antibiotics, which could lead to treatment failure clinically. Recently, the occurrence of ESBL-producing microorganism attracted wide concerns, which mainly existed in Escherichia coli(E. coli) and Klebsiella pneumoniae and wide distributed in hospital,community and animal breeding environments. The ESBLs mainly include three types, TEM,SHV and CTX-M, which are often located on mobile transfer elements. The ESBLs could disseminate with gene transfer and bacteria spread. With the use of antibiotics in animal breeding environments, more and more ESBL-producers occurred in the world. The animal original ESBL-producing bacteria, especially E. coli that widely existed in environments,animal guts and feces could spread through various routes to surrounding environments, and even enter into the food chain by animal products, which threatened public health.CTX-M is the predominant ESBL types in various environments. It mainly located on plasmids and could transfer with plasmid between bacteria to transmit the resistance gene.Conjugation is the main way for the spread of CTX-M types ESBLs, which have been widely detected.The main components of bentonite are SiO2 and Al2O3, largely produced in China.Bentonite is widely used for feed additives in poultry, pigs, fishes and other animals to prolong the staying time of the food in the gut, provide minerals and decrease the cost in China. Nano-materials shared the main components with bentonite has been reported to promote plasmid transfer. Thus whether the bentonite could promote the transfer of ESBL genes attracted wide concern. To learn the prevalence and dissemination of ESBL-producing E. coli in the pig breeding environments, and the effect of feed additive bentonite on ESBL gene transfer between bacteria and its mechanism, we conducted the following research.1. The prevalence and characterization of ESBL-producing E. coli in pig breeding environmentsTo learn the prevalence of ESBL-producing E. coli and its dissemination to the surrounding environments, we collected different kinds of samples including pig manure,indoor and outdoor air samples, water and sludge samples, and soil samples from 10 pig farms in Shandong regions. A total of 119 ESBL-producing E. coli was obtained from 6 pig farms of these samples. The other four pig farms were negative for ESBL-producers. CTX-Mand TEM, but not SHV were detected in these isolates. These isolates were resistant to at least two or three categories antibiotics. The results showed that the ESBL-producing E. coli from feces and environmental samples within the same farm had high association in resistance profile and similar CTX-M subtypes. These results suggested that the ESBL-producing E. coli was prevalent in this region and could disseminate to the surrounding environments.2. The dissemination of ESBL-producing E. coli from pig farms to surrounding soil due to manure application.ESBL-producing E. coli was used as an indicator to study the influence of animal original ESBL-producing E. coli on the planting soil amended with manure. From the feces,compost and soil samples collected from the farm and amended soil, a total of 42 ESBLproducing E. coli was isolated including 29 from fecal samples(isolation rate 72.5%), 3 from compost(15%) and 10 isolates from amended soil(12.5%). No ESBL-producers were obtained from the control soil treated with chemical fertilizer. The ESBL-producing isolates from pig farms and soil samples all showed multi-drug resistance. CTX-M and TEM were detected, and CTX-M-14 and CTX-M-15 were the predominant subtypes. IncF was the main replicon types, widely found in these isolates. Even several replicon types were co-existed in one isolates. ERIC-PCR results showed that 3 soil isolates had above 90% similarity with fecal isolates, indicating that manure application is a likely contributor of ESBL-producing bacteria to the soil.3. Study on the effect of feed additive bentonite on the transfer of CTX-M-15In the above part of this study, CTX-M-15 was detected to be the most predominant ESBL genes in the pig farms of this region. Thus we build a conjugation transfer model using the isolate from one pig farm carrying CTX-M-15 as donor bacteria, which was susceptible to kanamycin, and BL21(DE3) that able to resistant to kanamycin as recipient to study the effect of bentonite on the transfer of ESBL gene. The results showed that the CTX-M-15 gene could transfer between the donor and recipient strain, and bentonite could promote the transfer frequency of this gene to 4-20 times. The promotion effect was influenced by the concentration of bentonite.4. Comparative proteomics analysis of CTX-M-15 horizontal transfer under the condition of bentoniteIn this part, comparative proteomics was used to evaluate regulation proteins under the condition of bentonite to study the mechanism of the promotion effect of bentonite. Proteins were extracted from the bacteria mixture and subjected to two-dimensional(2D)electrophoresis to select the differential expressed proteins. 48 spots were differentialexpressed based on the selection condition that above 2 times change and p value below 0.05.29 proteins were identified using 5800 MALDI-TOF. Bioinformatics analysis showed that the proteins include 25 enzymes and 4 proteins involved in multiple and complex biological process including glycolsis, stress response, transport, protein biosynthesis, other biosynthesis, carbohydrate metabolism, glucose metabolism, antibiotic resistance, glycerol metabolism, biofilm formation and cell chemo taxis. The four proteins are outer membrane channel protein, maltose ABC transporter periplasmic protein, MltA-interacting protein MipA and ABC transporter ATP-binding proteinABC.5. Transcriptive level study of CTX-M-15 horizontal transfer under bentonite condition and the effect of four proteinsBased on the sequences of tolC, malE, mipA and ybi T on NCBI, primers of Real time fluorescence quantitative PCR and the whole open reading frame(ORF) of the four genes were designed respectively to study the change of transcription level. Quantitative PCR(qPCR) results showed that the expression level of RNA from donor, recipient and mixture strain were all up-regulated, consistent with changes in protein level. Furthermore, the regulation level of recipient was the highest, followed by the donor strains, which means that the recipient was susceptible to bentonite. Thus, we conducted the expression vectors of tolC,malE, mipA and ybiT and transformed them into BL21(DE3), which was used as recipients to evaluate the effect of the four proteins. The results showed that transfer frequencies of the four conjugation experiment were all increased.