水中产ESBL耐热大肠菌及耐药基因在小鼠肠道中的转归研究
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摘要
近年来,随着抗生素的大量运用,耐药菌的加剧出现成为严重影响人类健康的一个重要安全问题。产超广谱β内酰胺酶(Extended-spectrumβ-lactamases,ESBL)菌株1983年被Kliebe.C首次报道后,国际上对其的关注日益增多。产ESBL菌株在患者体内、畜牧、家禽、昆虫和环境中的不断检出,尤其是新型产ESBL菌株的不断发现,都提示产ESBL菌株业已成为当前的一类广泛流行的微生物,并已经和耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcus aureus, MRSA),耐万古霉素肠球菌(Vancomycin resistant Enterococcus,VRE)一并成为目前临床上威胁最大的三大菌株。产ESBL菌株主要为革兰氏阴性杆菌,大多数通过质粒介导。由于革兰氏阴性杆菌繁殖速度快,细菌群体效应密切,因此导致编码ESBL的质粒在菌株之间的转移相对阳性菌株更加容易,且发生变异的机会也更大。目前,世界上已经发现了近300多种编码ESBL基因(bla基因),这类菌株的耐药谱提示此类菌株的耐药类型几乎覆盖了目前所有的常用抗生素,其造成的感染会给临床治疗带来极大的困难。
水环境作为耐药基因传播的媒介,其庞大的耐药基因库,为进入该环境中的致病菌及条件致病菌提供获得大量耐药基因的机会,一旦这些致病菌再次入侵人体,引起感染,将会造成难以预计的后果。耐热大肠菌群作为水中致病菌的指示菌,具有易培养,易鉴别,数量众多的优点,可以客观地反映水中菌株的耐药情况。同时,由于耐热大肠菌株来源于温血动物的肠道,同肠道固有菌群具有遗传上的相似性,因此在细菌间交流对话和基因交换方面具有更加便利的条件,这样也会造成耐药型耐热大肠菌群成为耐药基因的储存库,通过基因转移将耐药基因传递到其他菌株,形成潜在的健康危害。本课题将从水中的产ESBL耐热大肠菌株为切入点,研究其在地表水中的分布情况,耐药特点及耐药相关基因元件的携带规律,并通过耐药基因的体内转归规律研究,探讨水中产ESBL菌株在水环境中的卫生学特点,以及此类菌株在进入人体后可能带来的健康危害,为控制水源性耐药菌的污染,预防耐药菌引起的介水传染病,保障公共卫生安全提供科学依据。
一、水中产ESBL耐热大肠菌群耐药谱及耐药基因型研究
我室前期已经发现,医院污水是地表水中的产ESBL菌株的主要来源,由于医院污水中的耐药菌具有致病菌多,耐药谱广的特点,我们推测,医院污水中产ESBL菌株在进入地表水体后,其所携带的耐药相关基因元件将会在阴性菌株中发生散播,造成耐药菌株的大范围扩散。因此,了解水中产ESBL菌株的菌种、耐药谱特点和产ESBL耐药基因型特点,对于掌握重庆地区长江地表水中产ESBL菌的污染水平,以及其可能造成的耐药性扩散危害,将提供十分重要的背景数据。
本实验从从长江地表水重庆市区段2公里的江面上,于2007至2009年分三次采集水样,分离水中耐热大肠菌群,通过产ESBL确证实验获得产ESBL耐热大肠菌,使用细菌菌种鉴定系统鉴定样本菌株菌种,测定对一线常用19种抗生素的MIC值,并使用特定的引物,PCR检测TEM、SHV、CTX-M和OXA四种产ESBL基因的携带情况。本实验共从重庆段长江地表水收集得到1914株水源性耐热大肠菌群,其中342株对头孢噻肟耐药,经过产ESBL确证实验共获得319株产ESBL菌株,占水中耐热大肠菌的16.7%。所有菌株均呈现多重耐药性,对19种一线抗生素有不同程度耐药,仅对阿米卡星和亚胺培南敏感率较高,通过分析样本菌株的bla基因型发现,CTX-M是水中产ESBL耐热大肠菌的主要的bla基因型,但是菌株以复合基因型为主要构成主体,同本地临床报道有较大差距。其中CTX-M+TEM占有较大比例(30.4%),其次是CTX-M+SHV(9.1%)和SHV+TEM(6.0%),未发现OXA基因型。结果显示,重庆段长江地表水中产ESBL菌株污染情况较为严重,呈现耐药谱广,耐药基因型复杂的特点,提示此类菌株在水中可能成为耐药基因的主要储存载体,地表水环境可能成为耐药基因重新组合的重要场所。
二、水中产ESBL耐热大肠菌群中整合子携带情况研究
整合子是细菌特别是革兰阴性菌基因中,一种能识别且俘获移动性基因盒,并有位点特异性的重组表达系统的可移动基因元件(mobile gene elements,MGEs)。整合子本身无法移动,但常出现在转座子,接合性质粒,噬菌体或细菌染色体上,共同作为可自行移动的载体,捕获和整合耐药基因,形成巨大的多基因座(loci) ,并随细菌的繁殖复制到子代DNA中。其所携带的基因盒可以编码对多种抗生素的耐药性,因此整合子被认为是同菌株多重耐药密切相关的一类MGEs。目前已知的整合子可以分为1、2、3、4类,其中1类整合子被认为在耐药基因的转移过程中占主要作用。本课题在前期发现产ESBL菌株呈现高度的多重耐药趋势的基础上,结合此类菌株对非产β-内酰胺类抗生素也具有较高的的耐药比例的特征,推测水中产ESBL菌株可能和整合子有着密切的联系,因此研究水中产ESBL菌株中整合子的的携带规律,对于掌握产ESBL菌株多重耐药的原因和特点,摸清此类菌株的耐药规律,揭示其可能产生的卫生学危害,具有重要意义。
通过对产ESBL耐热大肠菌进行1、2、3类整合酶扩增,并扩增和测序分析整合酶阳性菌株中耐药基因盒区域,发现319株样本菌株中,含有1类整合子比例较高,达65.2%。仅有3株菌株携带有2类整合子,未发现3类整合子的存在。3株携带2类整合子的菌株同时也携带1类整合子。通过分析整合子包含的耐药基因盒区域发现,208株整合酶阳性菌株中,共有117株整合子阳性菌株携带有耐药基因盒区域,具有9种不同的片段长度,其余菌株未检测到扩增产物。通过对扩增产物进行测序分析共检测到13种耐药基因盒,其中aadA家族和dfrA家族是产ESBL菌株中最常见的耐药基因盒,1.8kb片段中dfrA17-aadA5的耐药基因盒组合形式出现频率最高,且序列片段大小相对固定。结果提示,1类整合子在产ESBL菌株中存在几率较高,整合子中携带的耐药基因种类广泛,可能是造成此类菌株的多重耐药性的重要原因。耐药基因盒区域的相对固定表明,整合子在产ESBL菌株中可能作为一个保守的固有元件,进行整体传播,形成耐药性的扩散,整合子的保守性可能使其成为储存耐药基因的重要形式。
三、水源性产ESBL菌株在小鼠肠道内的定植情况研究
水中产ESBL耐热大肠菌群由于其分布广泛,且通过前期研究证明对氯制剂具有一定的抗性,常规的消毒方式无法完全杀灭水中的产ESBL菌株,可能通过饮水进入到人体肠道当中。此外,在一些容易接触到污水的职业人群,如污水处理厂工人、渔民等,以及卫生条件较差的农村地区,通过消化道感染此类菌株的机会更大。耐热大肠菌群来源于动物肠道,同肠道固有菌和致病菌之间都有着密切的遗传背景联系。当此类菌株进入人体肠道后,可能在肠道中定植下来,使得人体肠道菌群成为耐药菌的保存宿主,并可能通过耐药基因的扩散造成更为严重的健康危害,形成远期的健康威胁。因此,研究水源性产ESBL耐热大肠菌在肠道内的定植的规律,对于防控耐药菌污染,预防肠道菌群失调,有着十分重要的卫生学意义。
本课题中,我们通过模拟相当于清洁泉水、长江地表水、和医院污水水体中产ESBL菌株浓度的饮水,使小鼠自由饮用,用含头孢噻肟的m-FC培养基进行粪便中耐药菌株的检测和鉴定。研究发现,当饮水中产ESBL菌浓度相当于长江地表水水平时,小鼠正常接触该饮水6h,即可在粪便中检测到耐药菌的存在,说明在该浓度下接触污染饮用水,耐药菌可以顺利通过小鼠肠道并排出体外。在停止接触污染饮用水3d后,小鼠粪便中仍能继续检测到耐药菌的存在。各浓度饮水组小鼠在停止接触细菌后,粪便中耐药菌检出率逐步趋同,维持在103 CFU/g粪便水平,可稳定排出一定时间。通过RAPD方法对检出菌株进行分析,发现粪便中的耐药菌既存在饮水中存在的污染菌,又有获得耐药基因而产生耐药性的肠道固有菌菌,因此可以说明外源性产ESBL菌株在进入小鼠肠道内后,可以在小鼠肠道内定植下来,并提示肠道固有菌群通过捕获外源性细菌的质粒,获得其所携带的耐药基因,成为新的产ESBL耐药菌株。由于固有菌株在肠道内可以长期稳定的存在,因此可能造成肠道内固有菌群的耐药性提高,当受到抗生素选择压力时,造成肠道内菌群结构的改变,带来一定的健康危害。同时,外源性耐药基因可能通过在肠道内固有菌的存量作用,形成基因的扩散和复制,排除体外后引起更为广泛的耐药基因污染。研究结果显示,水源性产ESBL耐热大肠菌进入动物肠道后可以持续复制并通过粪便排出体外,肠道环境对于耐药菌具有储存和放大的作用。此发现提示,水源性产ESBL耐热大肠菌对于人体肠道存在一定的健康危害,需要加强对饮水安全的监控,提高饮水中耐药菌的杀灭效果,防止介水传染病的发生。
四、用于研究耐药基因肠道转移的新型小鼠模型的建立
耐药基因在肠道内基因转移一直是科学家们研究的重点,但是由于肠道菌群的种类繁多,菌落数量难以计算,使得肠道菌群内耐药菌相关实验的设计成为一个难以突破的瓶颈。在体外实验中,研究人员已经成功地实现了不同菌株、菌种甚至菌属之间的基因转移,但是在肠道内如何将此类研究变得可控化、可视化一直难以解决。本课题受到Babic发表于08年SCIENCE[1]上的文章启发,尝试从直接可视化层面对肠道内目标菌株进行改造,使得肠道内固有菌株在稳定生长的前提下,具有简单易行的可视化检测方法,结合抗生素耐药性可以快速筛选出捕获有外源性耐药基因的接合菌株,从而方便与计算和评价外源性耐药基因对肠道固有菌的影响作用。
我们通过将编码绿色荧光蛋白的gfp基因从pGFPuv质粒中提取出来,然后连接到pET28a+原核表达载体当中,构建成重组质粒pET28aGFP质粒,并将此质粒通过转染ArcticExpress感受态菌株,证实此重组质粒能够在宿主菌株中稳定表达绿色荧光。然后从小鼠肠道各肠段提取优势固有耐热大肠菌株,根据实验需要选择合适的菌种和耐药谱,然后通过基因重组将我们构建好的重组质粒整合到固有菌株当中,再通过饲喂菌株使得重组菌株在小鼠肠道内定植。通过检测粪便中发光菌的数量发现,我们构建的此种小鼠模型能够稳定从体内稳定排出发光重组受体菌株,并持续排出7d以上,可以说明构建的此种小鼠模型能够用于肠道菌株耐药基因的转移研究,为后续的耐药基因体内转移研究提供了较为实用的实验平台。
五、水源性产ESBL菌株所携带耐药基因在小鼠模型内的转移规律研究
以往的报道显示,对于小鼠肠道内耐药基因转移的研究,更多的是通过检测粪便中耐药菌的存在来推断,而缺乏对整个肠道系统而具体的了解,其转移频率也因为区分接合菌株困难而在统计上存在一定难度。我们在前期成功构建小鼠肠道菌群模型的基础上,利用各个肠段的优势菌群指示菌株作为受体菌,研究当水源性产ESBL耐热大肠菌进入小鼠肠道后,所携带的耐药基因想指示菌株转移的规律,对于揭示肠道内环境对于耐药基因转移的影响,以及外源性耐药基因在肠道中的转归提供了一定的实验依据。
研究发现,当水源性产ESBL菌株进入肠道后,在部分肠段内发生了耐药基因的转移。但是在胃、十二指肠和空肠上段中,均未能检测到耐药基因转移现象,说明酸性环境可能对耐药基因转移有一定抑制作用。肠道耐药基因转移在结肠内检出频率较高,且各类型产ESBL菌株之间无明显差异,说明结肠环境由于菌群密集,菌株数量多,菌体停留时间长,使得耐药基因的转移几率增大,甚至高于体外转移水平,提示肠道菌群中最有可能受到外源性耐药基因影响的菌群集中在结肠环境内,当肠道菌群受到抗生素影响时,可能会引起结肠的肠道菌群失调。分子生物学证据表明,获得了耐药基因的受体菌株同造模中使用的受体菌株具有同样的基因型,证明了(1)前期建立的小鼠模型能够成功运用于产ESBL菌株的肠道转移实验当中,具有良好的操作性;(2)水源性产ESBL菌株在进入肠道后,能够将所携带耐药基因转移给肠道固有菌,形成耐药基因的扩散,并可能通过肠道固有菌对耐药基因的保存,形成稳定持续的耐药基因生产场所。
六、结论和创新点
长江作为重庆的主干水流,已经受到产ESBL耐药菌的严重污染,水中耐药菌的检出水平远远高于其他地区。产ESBL耐热大肠菌呈现高度的多重耐药特点,对于目前临床和畜牧业上常用的一线抗生素均有不同程度的耐药,其中整合子可能在多重耐药性上发挥了重要作用。通过体内实验我们证实,水中产ESBL耐热大肠菌由于其适应肠道生活环境,且同肠道菌群之间关系密切,因此在进入动物肠道后能够定植并繁殖,并通过粪便持续性排出体外。同时,水源性产ESBL菌株还可以将所携带的耐药基因转移给肠道固有菌,形成耐药菌种类扩散,结肠环境是发生基因转移的重要场所。本课题提示,需要严格控制污染源中耐药菌的排放水平,加强对地表水中耐药菌的监控力度,进一步加强对饮水安全的措施保障,为防止耐药菌在水环境中的扩散,预防介水传染病的发生提供有力支持。
重庆作为一个长江上游地区发达的综合性城市,其耐药菌的产生和排放严重影响长江的水质。长江为重庆城区的主要饮水来源,且是下游三峡库区的重要主干水流,长江水中的菌株耐药情况同人群的健康关系密切。本课题在目前国内首次关注长江地表水中产ESBL耐热大肠菌的背景资料,并通过耐药谱、整合子、肠道定植、耐药基因转移等实验,从多个方面客观阐述了此类菌株在水中的分布特征及对动物肠道菌群的潜在危害,具有一定的现实意义。同时,本课题也是国内外率先通过重组肠道固有菌构建用于研究耐药基因体内转移的动物模型,并通过实验证实具有一定的应用效果,为耐药基因的相关研究提供了一定的科学依据和实验积累。
七、问题与展望:
本研究仅针对水源性耐热大肠菌群进行研究,而产ESBL菌株所涵盖的范围极广,不但包括了肠杆菌科,还包括了拟杆菌、假单胞菌等,因此仅仅研究肠杆菌科中耐热大肠菌群的产ESBL菌株特性还不能说明水中整体耐药菌株的特性。此外,我们在研究中发现,虽然临床报道表明2类整合子在临床分离株中携带比例也在一个较高水平,但是我们的样本菌株中仅发现3株菌株携带有2类整合子,提示耐热大肠菌株可能对于1类整合子有偏好,而较少的捕获2类整合子。因此,后期如果能对耐热大肠菌株对于1类整合子的捕获偏好机制进行研究,将有助于了解耐药基因捕获和转移的更深层次规律。同时,相比临床菌株,产ESBL菌株对1类整合子的捕获率明显较高,从菌株层面上考虑,是否此类菌株更容易识别外源性耐药基因,或者是否存在一定的分子机制使得此类菌株对于外源性耐药基因的整合和利用更加便捷,也是我们值得继续深入研究的一个重要方面。
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 TC
Our 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 coliforms
Integron 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 track
It 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 experiment
Although 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 vivo
The 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 Prospectives
This 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.
水环境作为耐药基因传播的媒介,其庞大的耐药基因库,为进入该环境中的致病菌及条件致病菌提供获得大量耐药基因的机会,一旦这些致病菌再次入侵人体,引起感染,将会造成难以预计的后果。耐热大肠菌群作为水中致病菌的指示菌,具有易培养,易鉴别,数量众多的优点,可以客观地反映水中菌株的耐药情况。同时,由于耐热大肠菌株来源于温血动物的肠道,同肠道固有菌群具有遗传上的相似性,因此在细菌间交流对话和基因交换方面具有更加便利的条件,这样也会造成耐药型耐热大肠菌群成为耐药基因的储存库,通过基因转移将耐药基因传递到其他菌株,形成潜在的健康危害。本课题将从水中的产ESBL耐热大肠菌株为切入点,研究其在地表水中的分布情况,耐药特点及耐药相关基因元件的携带规律,并通过耐药基因的体内转归规律研究,探讨水中产ESBL菌株在水环境中的卫生学特点,以及此类菌株在进入人体后可能带来的健康危害,为控制水源性耐药菌的污染,预防耐药菌引起的介水传染病,保障公共卫生安全提供科学依据。
一、水中产ESBL耐热大肠菌群耐药谱及耐药基因型研究
我室前期已经发现,医院污水是地表水中的产ESBL菌株的主要来源,由于医院污水中的耐药菌具有致病菌多,耐药谱广的特点,我们推测,医院污水中产ESBL菌株在进入地表水体后,其所携带的耐药相关基因元件将会在阴性菌株中发生散播,造成耐药菌株的大范围扩散。因此,了解水中产ESBL菌株的菌种、耐药谱特点和产ESBL耐药基因型特点,对于掌握重庆地区长江地表水中产ESBL菌的污染水平,以及其可能造成的耐药性扩散危害,将提供十分重要的背景数据。
本实验从从长江地表水重庆市区段2公里的江面上,于2007至2009年分三次采集水样,分离水中耐热大肠菌群,通过产ESBL确证实验获得产ESBL耐热大肠菌,使用细菌菌种鉴定系统鉴定样本菌株菌种,测定对一线常用19种抗生素的MIC值,并使用特定的引物,PCR检测TEM、SHV、CTX-M和OXA四种产ESBL基因的携带情况。本实验共从重庆段长江地表水收集得到1914株水源性耐热大肠菌群,其中342株对头孢噻肟耐药,经过产ESBL确证实验共获得319株产ESBL菌株,占水中耐热大肠菌的16.7%。所有菌株均呈现多重耐药性,对19种一线抗生素有不同程度耐药,仅对阿米卡星和亚胺培南敏感率较高,通过分析样本菌株的bla基因型发现,CTX-M是水中产ESBL耐热大肠菌的主要的bla基因型,但是菌株以复合基因型为主要构成主体,同本地临床报道有较大差距。其中CTX-M+TEM占有较大比例(30.4%),其次是CTX-M+SHV(9.1%)和SHV+TEM(6.0%),未发现OXA基因型。结果显示,重庆段长江地表水中产ESBL菌株污染情况较为严重,呈现耐药谱广,耐药基因型复杂的特点,提示此类菌株在水中可能成为耐药基因的主要储存载体,地表水环境可能成为耐药基因重新组合的重要场所。
二、水中产ESBL耐热大肠菌群中整合子携带情况研究
整合子是细菌特别是革兰阴性菌基因中,一种能识别且俘获移动性基因盒,并有位点特异性的重组表达系统的可移动基因元件(mobile gene elements,MGEs)。整合子本身无法移动,但常出现在转座子,接合性质粒,噬菌体或细菌染色体上,共同作为可自行移动的载体,捕获和整合耐药基因,形成巨大的多基因座(loci) ,并随细菌的繁殖复制到子代DNA中。其所携带的基因盒可以编码对多种抗生素的耐药性,因此整合子被认为是同菌株多重耐药密切相关的一类MGEs。目前已知的整合子可以分为1、2、3、4类,其中1类整合子被认为在耐药基因的转移过程中占主要作用。本课题在前期发现产ESBL菌株呈现高度的多重耐药趋势的基础上,结合此类菌株对非产β-内酰胺类抗生素也具有较高的的耐药比例的特征,推测水中产ESBL菌株可能和整合子有着密切的联系,因此研究水中产ESBL菌株中整合子的的携带规律,对于掌握产ESBL菌株多重耐药的原因和特点,摸清此类菌株的耐药规律,揭示其可能产生的卫生学危害,具有重要意义。
通过对产ESBL耐热大肠菌进行1、2、3类整合酶扩增,并扩增和测序分析整合酶阳性菌株中耐药基因盒区域,发现319株样本菌株中,含有1类整合子比例较高,达65.2%。仅有3株菌株携带有2类整合子,未发现3类整合子的存在。3株携带2类整合子的菌株同时也携带1类整合子。通过分析整合子包含的耐药基因盒区域发现,208株整合酶阳性菌株中,共有117株整合子阳性菌株携带有耐药基因盒区域,具有9种不同的片段长度,其余菌株未检测到扩增产物。通过对扩增产物进行测序分析共检测到13种耐药基因盒,其中aadA家族和dfrA家族是产ESBL菌株中最常见的耐药基因盒,1.8kb片段中dfrA17-aadA5的耐药基因盒组合形式出现频率最高,且序列片段大小相对固定。结果提示,1类整合子在产ESBL菌株中存在几率较高,整合子中携带的耐药基因种类广泛,可能是造成此类菌株的多重耐药性的重要原因。耐药基因盒区域的相对固定表明,整合子在产ESBL菌株中可能作为一个保守的固有元件,进行整体传播,形成耐药性的扩散,整合子的保守性可能使其成为储存耐药基因的重要形式。
三、水源性产ESBL菌株在小鼠肠道内的定植情况研究
水中产ESBL耐热大肠菌群由于其分布广泛,且通过前期研究证明对氯制剂具有一定的抗性,常规的消毒方式无法完全杀灭水中的产ESBL菌株,可能通过饮水进入到人体肠道当中。此外,在一些容易接触到污水的职业人群,如污水处理厂工人、渔民等,以及卫生条件较差的农村地区,通过消化道感染此类菌株的机会更大。耐热大肠菌群来源于动物肠道,同肠道固有菌和致病菌之间都有着密切的遗传背景联系。当此类菌株进入人体肠道后,可能在肠道中定植下来,使得人体肠道菌群成为耐药菌的保存宿主,并可能通过耐药基因的扩散造成更为严重的健康危害,形成远期的健康威胁。因此,研究水源性产ESBL耐热大肠菌在肠道内的定植的规律,对于防控耐药菌污染,预防肠道菌群失调,有着十分重要的卫生学意义。
本课题中,我们通过模拟相当于清洁泉水、长江地表水、和医院污水水体中产ESBL菌株浓度的饮水,使小鼠自由饮用,用含头孢噻肟的m-FC培养基进行粪便中耐药菌株的检测和鉴定。研究发现,当饮水中产ESBL菌浓度相当于长江地表水水平时,小鼠正常接触该饮水6h,即可在粪便中检测到耐药菌的存在,说明在该浓度下接触污染饮用水,耐药菌可以顺利通过小鼠肠道并排出体外。在停止接触污染饮用水3d后,小鼠粪便中仍能继续检测到耐药菌的存在。各浓度饮水组小鼠在停止接触细菌后,粪便中耐药菌检出率逐步趋同,维持在103 CFU/g粪便水平,可稳定排出一定时间。通过RAPD方法对检出菌株进行分析,发现粪便中的耐药菌既存在饮水中存在的污染菌,又有获得耐药基因而产生耐药性的肠道固有菌菌,因此可以说明外源性产ESBL菌株在进入小鼠肠道内后,可以在小鼠肠道内定植下来,并提示肠道固有菌群通过捕获外源性细菌的质粒,获得其所携带的耐药基因,成为新的产ESBL耐药菌株。由于固有菌株在肠道内可以长期稳定的存在,因此可能造成肠道内固有菌群的耐药性提高,当受到抗生素选择压力时,造成肠道内菌群结构的改变,带来一定的健康危害。同时,外源性耐药基因可能通过在肠道内固有菌的存量作用,形成基因的扩散和复制,排除体外后引起更为广泛的耐药基因污染。研究结果显示,水源性产ESBL耐热大肠菌进入动物肠道后可以持续复制并通过粪便排出体外,肠道环境对于耐药菌具有储存和放大的作用。此发现提示,水源性产ESBL耐热大肠菌对于人体肠道存在一定的健康危害,需要加强对饮水安全的监控,提高饮水中耐药菌的杀灭效果,防止介水传染病的发生。
四、用于研究耐药基因肠道转移的新型小鼠模型的建立
耐药基因在肠道内基因转移一直是科学家们研究的重点,但是由于肠道菌群的种类繁多,菌落数量难以计算,使得肠道菌群内耐药菌相关实验的设计成为一个难以突破的瓶颈。在体外实验中,研究人员已经成功地实现了不同菌株、菌种甚至菌属之间的基因转移,但是在肠道内如何将此类研究变得可控化、可视化一直难以解决。本课题受到Babic发表于08年SCIENCE[1]上的文章启发,尝试从直接可视化层面对肠道内目标菌株进行改造,使得肠道内固有菌株在稳定生长的前提下,具有简单易行的可视化检测方法,结合抗生素耐药性可以快速筛选出捕获有外源性耐药基因的接合菌株,从而方便与计算和评价外源性耐药基因对肠道固有菌的影响作用。
我们通过将编码绿色荧光蛋白的gfp基因从pGFPuv质粒中提取出来,然后连接到pET28a+原核表达载体当中,构建成重组质粒pET28aGFP质粒,并将此质粒通过转染ArcticExpress感受态菌株,证实此重组质粒能够在宿主菌株中稳定表达绿色荧光。然后从小鼠肠道各肠段提取优势固有耐热大肠菌株,根据实验需要选择合适的菌种和耐药谱,然后通过基因重组将我们构建好的重组质粒整合到固有菌株当中,再通过饲喂菌株使得重组菌株在小鼠肠道内定植。通过检测粪便中发光菌的数量发现,我们构建的此种小鼠模型能够稳定从体内稳定排出发光重组受体菌株,并持续排出7d以上,可以说明构建的此种小鼠模型能够用于肠道菌株耐药基因的转移研究,为后续的耐药基因体内转移研究提供了较为实用的实验平台。
五、水源性产ESBL菌株所携带耐药基因在小鼠模型内的转移规律研究
以往的报道显示,对于小鼠肠道内耐药基因转移的研究,更多的是通过检测粪便中耐药菌的存在来推断,而缺乏对整个肠道系统而具体的了解,其转移频率也因为区分接合菌株困难而在统计上存在一定难度。我们在前期成功构建小鼠肠道菌群模型的基础上,利用各个肠段的优势菌群指示菌株作为受体菌,研究当水源性产ESBL耐热大肠菌进入小鼠肠道后,所携带的耐药基因想指示菌株转移的规律,对于揭示肠道内环境对于耐药基因转移的影响,以及外源性耐药基因在肠道中的转归提供了一定的实验依据。
研究发现,当水源性产ESBL菌株进入肠道后,在部分肠段内发生了耐药基因的转移。但是在胃、十二指肠和空肠上段中,均未能检测到耐药基因转移现象,说明酸性环境可能对耐药基因转移有一定抑制作用。肠道耐药基因转移在结肠内检出频率较高,且各类型产ESBL菌株之间无明显差异,说明结肠环境由于菌群密集,菌株数量多,菌体停留时间长,使得耐药基因的转移几率增大,甚至高于体外转移水平,提示肠道菌群中最有可能受到外源性耐药基因影响的菌群集中在结肠环境内,当肠道菌群受到抗生素影响时,可能会引起结肠的肠道菌群失调。分子生物学证据表明,获得了耐药基因的受体菌株同造模中使用的受体菌株具有同样的基因型,证明了(1)前期建立的小鼠模型能够成功运用于产ESBL菌株的肠道转移实验当中,具有良好的操作性;(2)水源性产ESBL菌株在进入肠道后,能够将所携带耐药基因转移给肠道固有菌,形成耐药基因的扩散,并可能通过肠道固有菌对耐药基因的保存,形成稳定持续的耐药基因生产场所。
六、结论和创新点
长江作为重庆的主干水流,已经受到产ESBL耐药菌的严重污染,水中耐药菌的检出水平远远高于其他地区。产ESBL耐热大肠菌呈现高度的多重耐药特点,对于目前临床和畜牧业上常用的一线抗生素均有不同程度的耐药,其中整合子可能在多重耐药性上发挥了重要作用。通过体内实验我们证实,水中产ESBL耐热大肠菌由于其适应肠道生活环境,且同肠道菌群之间关系密切,因此在进入动物肠道后能够定植并繁殖,并通过粪便持续性排出体外。同时,水源性产ESBL菌株还可以将所携带的耐药基因转移给肠道固有菌,形成耐药菌种类扩散,结肠环境是发生基因转移的重要场所。本课题提示,需要严格控制污染源中耐药菌的排放水平,加强对地表水中耐药菌的监控力度,进一步加强对饮水安全的措施保障,为防止耐药菌在水环境中的扩散,预防介水传染病的发生提供有力支持。
重庆作为一个长江上游地区发达的综合性城市,其耐药菌的产生和排放严重影响长江的水质。长江为重庆城区的主要饮水来源,且是下游三峡库区的重要主干水流,长江水中的菌株耐药情况同人群的健康关系密切。本课题在目前国内首次关注长江地表水中产ESBL耐热大肠菌的背景资料,并通过耐药谱、整合子、肠道定植、耐药基因转移等实验,从多个方面客观阐述了此类菌株在水中的分布特征及对动物肠道菌群的潜在危害,具有一定的现实意义。同时,本课题也是国内外率先通过重组肠道固有菌构建用于研究耐药基因体内转移的动物模型,并通过实验证实具有一定的应用效果,为耐药基因的相关研究提供了一定的科学依据和实验积累。
七、问题与展望:
本研究仅针对水源性耐热大肠菌群进行研究,而产ESBL菌株所涵盖的范围极广,不但包括了肠杆菌科,还包括了拟杆菌、假单胞菌等,因此仅仅研究肠杆菌科中耐热大肠菌群的产ESBL菌株特性还不能说明水中整体耐药菌株的特性。此外,我们在研究中发现,虽然临床报道表明2类整合子在临床分离株中携带比例也在一个较高水平,但是我们的样本菌株中仅发现3株菌株携带有2类整合子,提示耐热大肠菌株可能对于1类整合子有偏好,而较少的捕获2类整合子。因此,后期如果能对耐热大肠菌株对于1类整合子的捕获偏好机制进行研究,将有助于了解耐药基因捕获和转移的更深层次规律。同时,相比临床菌株,产ESBL菌株对1类整合子的捕获率明显较高,从菌株层面上考虑,是否此类菌株更容易识别外源性耐药基因,或者是否存在一定的分子机制使得此类菌株对于外源性耐药基因的整合和利用更加便捷,也是我们值得继续深入研究的一个重要方面。
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 TC
Our 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 coliforms
Integron 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 track
It 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 experiment
Although 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 vivo
The 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 Prospectives
This 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.
引文
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