| Since ESBL-producing bacteria were first reported by Kliebe C in 1983, these kinds of strains have attracted many researchers'attentions. Nowadays, with the increasing usage of antibiotics, the emergence of antibiotic resistant bacteria have become a sever hygiene problem which could impact the health of human being. It suggested that ESBL-producing bacteria have turned to be the main kind of resistant strains with the reports about recovering in patients, poultry, livestock, and insects. It has become one of the three most important resistant bacteria, together with methicillin resistant Staphylococcus aureus (MASA), and vancomycin resistant Enterococcus (VRE). Gram-negative bacteria are the main part of ESBL-producing strains, which were mediated by plasmids. Because the fast reproduction speed of Gram-negative bacteria and close relation in quorum sensing system, it is more convenient to exchange resistance plasmid between strains, comparing the situation between gram-positive bacteria. From now on, more than 300 kinds of ESBL have been recovered, most of the strain who producing ESBL mediated multi drug resistance to the first line antibiotics. The infection caused by these kinds of strains will bring strait to clinic treatments.As the media of antibiotic resistance gene transfer, the bulky gene pool facilitated the resistance gene acquired by pathogens or conditional pathogens. Once these resistant bacteria invade into body and caused infection, the antibiotic used for treatment will be limited. As the indicator of faecal pollution, thermotolerant coliform (TC) are easy to be cultured and identified, so the resistance profile of TC could reflect the entire resistance situation in water bacteria. And more, TC only come from warm-blooded animals, which means there are similar to gut microflora at the genetic level and very easy to exchange gene, these make TC to be antibiotic resistance gene pool. In our study, taking the ESBL-producing TC as the entry point, we attend to study the resistant TC distribution pattern in water body and the gene transfer in animal gut, to evaluate the risk on animal health brought by water-borne ESBL-producing TC.Part I. The profile of antibiotic resistance and bla gene of water-borne ESBL-producing TCOur previous work demonstrated that there were plenty of ESBL-producing bacteria in Yangtze River surface water of Chongqing basin. By analyzing resistant strains origin from hospital wastewater and sewage, we found that the ESBL-producing strains from hospital wastewater are the main resource of surface water. Considering the traits of broad spectrum resistance and high dense of bacteria, we believed that the antibiotic resistance gene elements could spread among Gram-negative bacteria after entering surface water body, which could leading to the wide dissemination of antibiotic resistance. Therefore, it is very important to pay close attention to the species, resistance profile and resistance gene genotype of water-borne ESBL-producing bacteria.In our study, we totally obtained 1914 strains water-borne TC by water sample collecting. Among these isolates, 342 isolates were resistant to cefotaxime, and after double disk diffusion methods to confirm ESBL-producing, 319 isolates (319 of 1914, 16.7%) were verified to be ESBL-producing strains. All isolates were multi drug resistant, at least resistant to 19 kinds of first line antibiotics used in this study. All isolates were resistant to first and second generation cephalosporin, but showed different among the resistance refer to third generation cephalosporin. Except few isolates were resistant to amikaxin and imipenem, all strains presented high resistance prevalence (>50%), which suggested that this kind of strain have become a predominant carrier of antibiotic resistance genes. Through analyzing the bla gene type of ESBL-producing bacteria isolated in this study, we found that CTX-M gene type have constituted the largest portion of ESBL-producing gene types in Chongqing area. But quite a few isolates (158 strains in total, 49.5%) harbored more than one type ofβ-lactamase genes. blaTEM+CTX-M was detected in 97 isolates (30.4%), which constitute the largest portion of combined bla gene type. Comparing to the reports from the local hospital and other areas, it claimed the bla gene types in water-borne ESBL-producing bacteria were more complex, which means that the water environment have become a important space for the recombination of antibiotic resistance genes. Part II. Research on the integron and gene cassettes carried by water-borne ESBL-producing thermotolerant coliformsIntegron who was first reported by Stokes and Hall in 1989, were consider as an important mobile genetic elements which associated with multi drug resistance. Integron is the one in bacteria, especially Gram-negative baceria, who have the ability to capture mobile gene cassette and have a site-specific recombinant expression systems. Integron was not movable by itself, but often transfer between microorganism after integrating in transposon, conjugative plasmids, phages or bacteria chromosomes. With the host gene system, they could capture more than one kind of resistant genes to formed a loci, and transfer into offspring by reproduction. The various kinds of gene cassettes carried in integron could mediate multi antibiotic resistance. Now 4 kinds of integron have been reported, and class 1 integron was considered as an important role in the resistance gene transfer. Based on the finding of multi drug resistance trends in ESBL-producing bacteria, we found that there is much difference on resistance characteristic between beta-lactamase antibioitics and non-beta-lactamase antibiotics. So we believed that there must be close relationship between integron and ESBL-producing bacteria. So it is very important to study the situation of integron carried by ESBL-producing bacteria, in order to ascertain the reason of multi drug resistance.Of the 319 ESBL-producing isolates, 208 (65.2%) isolates carried class 1 integrons. Class 2 integrons were only detected in E.coli CQ069 and K. pneumoniae CQ133, CQ185 strains. No intI3 was detected in this study. The class 1 integron-carrying situation among different bla genes types is shown in Table 2.χ2 test was used to calculate the P-value of each bla gene type group, and no significant difference integron-carrying among different bla groups (p>0.05) was found.For the detection of integron cassettes (gene cassette regions) in all integron-positive isolates, 117 of 208 class 1 integron carrying isolates harbored gene cassettes while the rest showed'empty'. The sizes of class 1 integron gene cassette regions ranged from 0.2 to 3.2 kb. 1.8 kb amplicons are the most common gene cassette region harbored in class 1 integrons (36 of 117, 30.8%). 12 strains carried more than one gene cassette region, that is 0.2 kb + 2.2 kb in 3 isolates, amplicon 1.8 kb + 2.4 kb were detected in 4 isolates; 0.6 kb+2.2 kb in 5 isolates. Class 2 integron-positive isolates CQ069 harbored a 1.8 kb gene cassette region on intI1 and a 2.2 kb on intI 2, respectively. The results suggested that integron may be main reason of multi drug resistance which mediates the dissemination of resistance genes.Part III. The implanting situation of water-borne ESBL-producing strains in mice intestine trackIt is well known that the ESBL-producing bacteria appear in many environment, especially in water bodies. Considering the resistance to chlorine disinfectant, it should be paid attention on threaten to gut microflora after entering the intestinal track. The thermotolerant coliforms were origin from mammalian animals'intestinal environment, which were similar to gut commensal bacteria on the genetic background, and made it is easy to transfer ESBL-encoding plasmid they carried to the gut commensal strains. This may lead a potential healthy harm. In view of this situation, it very meaningful to study the appearance of ESBL-producing strains implanting in the host gut environment through intake polluted drinking water, and to evaluate the threaten these strains brought to the host.We detected the resistant coliform from the feces of mice after intaking drinking water polluted by ESBL-producing coliform. The water types were designed to imitate the different water body which could be divided by the ESBL-producing coliform concentration level. When the concentration of ESBL-producing stains comes to meet the level of surface water of Yangtze River, the resistant strain could be detected 6h after the mice drunk the polluted water, it suggested that not only could the strains survive in the gut of mice, but also the strains could be excreted by the host. When the exposure to polluted drinking water were stopped for 3 days, resistant strains could also be recovered from feces, the results acquired by molecular analyze methods prompted that there might be a transconjugation occurred between the intaken strains and gut commensal bacteria.Part IV, the construction of a novel mice model using for the horizontal gene transfer experimentAlthough many scientists were trying hard to study the gene transfer in gut,it is still difficult to be achieved. The missing information of gut microflora is the very important key which is hard to be solved. Though in vitro experiment, the transfer of resistance gene between different strains, species, and even different genus were complied. But how to visualize the gene transfer in gut and avoid false positive results were not solved yet. Inspired by the reports published in Science, 2009, from Ana Babic, we intended to modify a kind of mice model which the gut flora could be selected and visualized by eyes. The biochemistry profiles of the strains were not changed apparently, and the conjugatant were well to be selected when using antibiotics selective medium.Firstly we obtained a gfp gene which encoding green fluorescent protein from vector pGFPuv, and ligated it to a prokaryotic expression vector pET28a+. The recombinant plasmid was transfer to ArcticExpressTM DE3 component cell to verified the function of green fluorescent. Then we got the indigenous thermotolerant coliform from the different parts of mice intestinal track, and transferred the recombinant plasmid to these strains to form recombinant ones, and administration of recombinant strains to mice were performed. By detecting the photobacterium in feces of mice, it demonstrated that the recombinant strains could implant in the gut of mice and be expelled out of body at least in 6 days. A longer detectable time was not taken but it is still enough to prove that the novel mice model were made successfully and it is could be applied in the continuing experiments.Part V. the study of bla gene transfer from water-borne ESBL-producing coliform to mice model in vivoThe previously reports demonstrated that most of the researches about the transfer of resistance gene in mice gut were performed by detecting the resistant strains in feces, which is lack of the concise sight on the entire intestinal track. It is also difficult to calculate the transconjugation frequencies for the inconvenient of distinguish the strains in feces. Based on the novel mice model we made in previous work, the gut commensal strains were used as the recipients to study the bla gene transfer from ESBL-producing strains to recipients. It is meaningful to gain the relationship between exogenous resistant strains and gut commensal microflora.The results indicates that when the water-borne ESBL-producing strain entered the gut of mice model, the gene transfer have taken places in some parts of intestinal track. We could not observe the transfer in stomach, duodenum, and upper jejunum, which suggested that the acidic environment could inhibit the transfer between strains. Higher transfer frequencies were observed in colon proved that the colon environment is an ideal place for the gene transfer. It also gives us a alarm that when the microflora in gut were treated by antibiotics, the constitution could be changed and lead to dysbacteriosis. The molecular evidences proved that:(1) the mice model could be used in the transfer experiments and work well as we expected; (2) when the water-borne ESBL-producing strains entered the gut, the resistance gene could be transferred to the gut commensal flora, which could lead to the spread of resistance in strains. (3) With the preservation of resistance gene, the gut could be turned to be a place where the resistance gene could be duplicated and expelled continuously.Conclusion and Innovation:As the main water body of Chongqing city, Yangtze River has been polluted by ESBL-producing bacteria heavily, the level of resistant bacteria was much higher than the other areas. ESBL-producing strains were multi drug resistance, which showed resistant to the first line antibiotics common used in clinic and animal products, among this, integron may play an important role in multi drug resistance. The in vivo experiments demonstrated that ESBL-producing bacteria could implant presistantly in gut environment for their adaptance in intestinal and close relationship with gut commensal microflora. Meanwhile, the gene transfer could be observed from water-borne ESBL-producing bacteria to gut commensal bacteria, leading to the dissemination of antibiotic resistance spread. The colon is tne main spaces for this transfer. Our study suggersted that it is very important to control the level of resistant bacteria expelling, and strengthen efforts to monitor the resistant bacteria in surface water, besides, the drinking water safety should be further guaranteed, in order to prevent the widespread of resistant bacteria in water environment and reduce the occurrence probability of water-boren infectious diseases.Chonqqing is a well developed city in the upstream of Yangtze River, the resistant bacteria produced in this city directly affected the water safety of Yangtze River. But so far, the characteristion of antibiotic resistance of water-borne bacteria in Yangtze River have not been reported. We were the first study concerning about the background of resistant bacteria in Yangtze River, and we performed the experiments about profile of antibiotic resistance, integron, implantion in gut, and gene transfer in vivio, to demonstrated the distribution in water environment and the healthy harzards of this kind of strains, which process the practice signification meaning. Meanwhile, this is the first attampt of study gene transfer using recombintant gut commensal recipient, the results demonstrated that this mice model have some practical foreground in studying the in vivo gene transfer. Problems and ProspectivesThis study was only focused on the water-borne ESBL-producing coliform, as we know, not only coliform, but also are many kinds of strains, such like bacteroides, pseudomonas, involved in these kind of strains. So it is not enough to explain the whole situation of ESBL-producing strains in water body. And more, the clinic reports claimed that class 2 integron were carried at a high rate in clinic isolates, but we only found 3 strains carried this kind of integron, which suggesting that there would be a trends for class 1 integron in ESBL-producing coliforms. Comparing to the clinic isolates, the carrying rate of class 1 integron were significant high, it reminded us to take a deep sight at the captured mechanism of integron integrating in these kinds of strains, which could be useful for the further understanding of gene transfer.
|
PDF Full Text Request