Amplification And Transfer Of Antibiotic Multiresistance Plasmids In Zebrafish Guts

Background:The pollution of antibiotic resistance bacteria (ARB) and the dissemination of antibiotic resistance genes (ARGs) have become global crises. The World Health Organization (WHO) has reported that the antibiotic resistance bacteria are spreading all over the world. Until 2050, antibiotic resistance will cause premature death of 1 million people each year. The issue of ARGs spread is so severe that they have been listed as an emerging type of environmental pollutants, which has become the focus and hotspot of international research. The majority of ARGs are encoded on the plasmids. It is the most common and effective way to transfer and disseminate ARGs between microorganisms via plasmid-mediated conjugative transfer.The surface water is a huge reservoir for ARB and ARGs. It not only collects and accommodates innumerable and various ARB, ARGs even multidrug resistance genes from different types of water, such as rainfall runoff, hospital effluents, aquaculture wastewater, laboratory sewage and so on, but also carries parts of the endogenous pollution, for example, the randomly resistant mutations and natural ARGs transfer in water environment. Aquatic animals, that live in the environment filled with various ARGs, may ingest ARGs, or antibiotic resistance bacteria (ARB), into their intestines where many microorganisms reside. Aquatic animal gut is a suitable niche for the growth and proliferation of bacteria, meanwhile, the large number of indigenous gut flora are able to serve as the potential recipient in horizontal gene transfer. Therefore, the transfer and dissemination of ARGs in aquatic animal guts may also be parts of the endogenous ARG contamination of surface water. Because the efficiency of ARG mutation and natural transfer is at the quite low level of only one in a million, and it is found that the self-purification of water, the presence of variously physical, chemical and biological factors especially the existence of abundant DNA enzymes even did not lead to the denaturation and decomposition of ARGs. The pollution of ARB and ARGs in water is still serious. In addition, there have been increasing reports demonstrated that multiple ARGs have been detected in aquaculture water and the feces and intestines of fish. Based on all the facts above, we believe that except for receiving ARGs in various water sources, as well as ARG mutation and randomly natural transfer, more importantly, the ARGs in water environment are likely to come from the amplification and dissemination of ARGs in aquatic animal guts. Thus, we proposed the hypothesis that the aquatic animal guts were important niche for the transfer and amplification of ARGs in the surface water.Purpose and significance:The purpose of this study is to investigate the changing regulations for ARB colonization and ARGs transfer qualitatively and quantitatively, explore the potential mechanisms of ARG transfer and dissemination and indicate the intestinal region where ARB colonization and ARG transfer most frequently occurred. Also we want to find out that how ARG transfer in zebrafish took place and which types of recipients obtained plasmids most readily by establishing RP4-mediated ARG transfer models in zebrafish gut. Our research will further enrich our knowledge of ARG transfer and dissemination in aquatic animal guts, and provide the theoretical and technical support for using antibiotics in aquaculture, management of water pollution and ecological environmental protection.Contents and methods:(1)We fed the zebrafish with donors K12Cm:RP4 which was established by RED recombination technology, and constructed the RP4-mediated conjugative transfer models in zebrafish gut.(2)We researched on the changing regulations of ARB as well as ARGs in the excretion of zebrafish over time by cultivation method associated with qPCR.(3)Research on the spatial and temporal distribution of ARB and ARGs in zebrafish gut by cultivation method associated with qPCR.(4)Identification the compositions and abundances of transconjugant species in zebrafish feces and guts by using dual-fluorescence labeling techniques, flow cytometry sorting and 16S rDNA amplicon sequencing.(5) Monitoring the mRNA expression of regulatory genes on RP4 plasmid in zebrafish guts using qPCR techniques.Results:(1)Zebrafish gut indigenous bacteria did not harbor RP4 plasmids, and were sensitive to Tc, Km and Cm but resistant to Amp. On the basis that none of the zebrafish was colonized with chloramphenicol-resistant bacteria and RP4 plasmids. We constructed the donors K12Cm:RP4 on the basis of the wild type E. coli K12 by using RED recombinant technology The donors with the genome marked by cat gene, harbored RP4 plasmid. We established the conjugative transfer models in zebrafish gut by constantly feeding animals donors mixed with feeds at fixed time during rationing.(2)The exogenous ARB and ARGs ingested by zebrafish tended to be deposited in large numbers in excreted feces. 12% of the bacteria in fish feces obtained ARGs through horizontal gene transfer mediated by RP4 plasmid. There was a change in bacterial dynamics in the feces of fish that could clearly be divided into three stages: the rise phase,the plateau phase and the descent phase. The high-dose group in which animals were ingested with the donors of 108cfu/(d·fish), the relative quantity of cat and traG gene was excreted with feces at the level of 2.37% and 15%, respectively. While the mid-dose group in which animals were ingested with donors of 107cfu/(d·fish), the relative quantity of cat and traG gene was excreted with feces at the level of less than 1%. It took a shorter time for the donors and transconjugants to reach the plateau phase in the mid-dose group, and the number of tranconjugants recovered was lower than almost a magnitude order compared with the high-dose group. As for the low-dose group in which the animals were ingested with donors of 106cfu/(d·fish),there was no clear change regulations of ARB and ARGs, transconjugants were also hardly detected, which suggested that generations of transconjugants and the time to reach the plateau was closely related to the initial feeding concentrations of donors.(3)The process of colonization and proliferation of ARB in vivo did not occur homogeneously throughout the gut but is rather preferentially localized to the hindgut region No significant difference was observed between the foregut and midgut regarding ARB and ARGs. The hindgut was the most important intestinal region for ARG transfer and dissemination, with concentrations of transconjugant cells almost 25 times higher than those of other intestinal segments. The donors and transconjugants was only transient because when the introduction of donors to zebrafish guts was stopped, both donors and transconjugants rapidly decreased in a very short period of time. The potential reasons were that our research was carried out without the selective pressure of antibiotics. However, transfer would take place in vivo whenever the aquatic animals and the ARB-harboring plasmids were in contact.(4)We also demonstrated that the transconjugants comprised both cultivable and uncultivable populations, and the uncultivable bacteria including Aeromonas,Polynucleobacter and Cetobacterium accounted for 90.4-97.2% of bacteria. Furthermore,we demonstrated that cultivable bacteria only accounted for 2.8-9.6% of bacteria.(5)The transfer and amplification of antibiotic resistance plasmids were closely related to the mRNA expression of regulatory genes in zebrafish hindgut. At the plateau phase, the mRNA expression levels of trbBp were significantly up-regulated in hindgut and reached 7.16×10-3 copies/cell which was 24 times the level observed at rise phase.The up-regulation of mRNA expression of trbBp gene impelled the mating pairs between cells, thus forming the joint channels, which promoted the first step of conjugation.Meanwhile, the mRNA expression of trfAp gene was significantly increased to 2.42×10-2 copies/cell, which impelled the transmembrane transport of RP4 plasmids, as well as the replication, transfer and distribution of plasmid DNA, thus promoting the second process of conjugation. However, the mRNA expressions of both regulatory genes were not significantly up-regulated. Therefore, the hindgut was the important intestine region where conjugative transfer occurred most frequently and active.Conclusions:(1)The establishment of stable RP4-mediated ARG transfer and dissemination models in zebrafish gut.(2)The majority of exogenous ARB were excreted and deposited in the feces. 12% of the fecal bacteria obtained RP4 plasmids by conjugation. After feeding the zebrafish donors for a period of time then stopped, the curve of ARB and ARGs in feces could be divided into three stages: the rise phase, the plateau phase and the descent phase.(3)The hindgut is the most important intestinal region for ARB colonization and ARG transfer.(4)The majority of recipients in zebrafish gut were uncultivable bacteria (accounted for 90.4%-97.2%) among which Aeromonas (>90%) was the most abundant bacterial genus to obtain RP4 plasmids. The cultivable bacteria only accounted for less than 10%, mainly including Enterobacteriaceae and Shewanella.(5) The considerably amplification and dissemination of ARGs in zebrafish hindgut was closely related to the up-regulation mRNA expression. The hindgut was the intestinal regions where conjugative transfer occurred most frequently and active.