【作者】 朱静媛；

【导师】 段广才；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2010， 博士

【摘要】 志贺菌属(Shigella)是感染性腹泻的重要病原,它引起的细菌性痢疾(菌痢)发病率在我国居传染病发病率的前三位,是我国传染病防治工作的重点。抗生素应用于临床治疗菌痢历史已久,不仅可以减轻病情、缩短病程,还能减少病人排菌,避免进一步传播。然而随着抗生素的广泛使用,临床频繁出现耐药志贺菌属菌株,而且呈现为多重耐药(multi-drug resistance, MDR)。多重耐药志贺菌引起的感染不仅在发展中国家,在发达国家也成为影响临床治疗的关键因素。多重耐药志贺菌属不仅给细菌性痢疾的防治带来新的挑战,对细菌耐药性的传播也产生重要影响。因此,志贺菌属细菌的多重耐药问题和耐药机制引起了人们的广泛关注。志贺菌属获得某种抗生素抗性主要通过两种机制,一是细菌自身染色体基因发生突变,如抗生素靶位点的突变、调控基因突变引起膜蛋白表达的异常或主动流出泵系统的激活等,使细菌对抗生素从敏感变成抗性；另一机制则是细菌从外部获得耐药基因,即耐药基因的水平转移,这是目前人们认为临床多重耐药株产生的主要原因。许多可移动基因单元如质粒、转座子等,携带抗生素耐药基因,使得耐药基因在细菌属、种、株间传播,加快了耐药菌株的形成。近年发现整合子系统参与耐药基因的传播。整合子包括整合酶、基因盒和特异性重组位点,整合酶可以不断地从周围环境捕获耐药基因盒或从整合子上切除基因盒。整合子作为一种遗传元件,它自己不能自由移动,但它可以位于质粒、转座子或者染色体上,通过其他可移动元件,整合子促进了耐药基因的扩散,自身的广泛分布与肠道菌多重耐药性的形成有关。研究表明,含有整合子的细菌菌株比不含有整合子的菌株更多的显示出对不同抗生素的耐药性,整合子—基因盒系统与志贺菌属多重耐药性有关。国内外对志贺菌属整合子的研究发现多重耐药志贺菌携带2类整合子多见,而1类整合子检出率相对较低。对于耐药基因盒类型而言,在志贺菌属中发现的类型相对较单一。不论整合子的类型还是携带耐药基因盒的种类,都与志贺菌属菌株的多重耐药表型没有一一对应关系。因此,探索整合子-耐药基因盒系统与志贺菌属多重耐药的关系、评价其在志贺菌属细菌多重耐药机制中的作用和地位是十分必要的。本研究对临床分离的83株福氏志贺菌和7株宋内志贺菌进行抗生素敏感性测定,比较不同类型整合子及耐药基因盒在志贺菌属的分布、构成差异,并对典型整合子-耐药基因盒系统进行克隆和功能分析,以期了解整合子-耐药基因盒系统与志贺菌属多重耐药性之间的关系、正确评价整合子-耐药基因盒系统在志贺菌属多重耐药机制中的作用和地位。方法1药敏实验用纸片扩散法(disc diffusion test)测定所有菌株对以下抗生素药敏纸片的抑菌环：氨苄青霉素(AMP)、四环素(TET)、甲氧苄啶/磺胺甲噁唑(SXT)、氯霉素(CHL)、萘啶酸(NAL)、环丙沙星(CIP)、庆大霉素(GEN)和头孢唑林(CFZ)。采用琼脂稀释法测定以下抗生素的最低抑菌浓度(minimum inhibition concentration, MIC):TET、AMP、CHL、CIP、链霉素(STR)、甲氧苄啶(TMP)和磺胺异噁唑(SSS)。2 PCR方法检测整合子的整合酶基因PCR-RFLP分析整合子的可变区基因分别针对1、2、3类整合子的整合酶基因设计引物,煮沸法提取菌株总DNA进行PCR扩增检测整合酶基因。用试剂盒分别提取菌株的基因组DNA和质粒DNA,分别用针对典型1类整合子的可变区引物hep58-hep59.针对非典型1类整合子的可变区引物hep58-ISVR、针对2类整合子的可变区引物hep74-hep51,进行整合子的可变区基因PCR扩增检测。选用合适的限制性内切酶,对得到相同长度大小的PCR产物进行RFLP (restrictive fragment length polimorphism)分析,得到相同内切酶图谱的片段被认为是序列相同的片段。3 Southern杂交按Roche公司地高辛标记和检测试剂盒(Dig DNA Labeling and Dection)的操作说明书进行操作。首先将整合酶基因intI 1和intI 2的PCR产物经凝胶回收纯化后定量,用随机引物法获得地高辛标记探针。将提取的细菌基因组DNA分用BamHI、PstI双酶切,将酶切产物和质粒DNA分别进行0.8%琼脂糖凝胶电泳。用毛细管虹吸法将琼脂糖凝胶中的DNA转移至尼龙膜,120℃烤箱干烤0.5h,将DNA固定于尼龙膜。经过预杂交、探针杂交、洗膜、避光显色等步骤,照相记录结果。4质粒接合传递实验以E. coli DH5a空菌为受体,以90株志贺菌为供体分别进行。分别挑取纯化的供体菌、受体菌单个菌落用LB液体培养基37℃振荡培养过夜,10倍稀释入2 ml新鲜的LB液体培养基,连续37℃振荡培养1-3 h使得OD600值约为0.6左右。吸取供体菌、受体菌各100μl于1 ml的新鲜LB液体培养基中,于37℃静止培养16-18h,划线接种于麦康凯固体培养基平板,37℃恒温培养箱内培养16-18h。观察菌落形态,在无色半透明菌落中夹杂的红色菌落即为接合子,挑取单一的红色菌落接种LB液体培养基,37℃振荡培养过夜。再次划线接种麦康凯固体培养基平板,37℃恒温培养箱内培养16-18h后观察形态以再次确认。提取质粒DNA,进行琼脂糖水平凝胶电泳检测。对获得确认的接合子进行纸片法药敏试验。5整合子-耐药盒基因的序列测定和系统发生分析针对典型1类整合子整合酶和可变区整个区域、非典型1类整合子的可变区区域、2类整合子整合酶和可变区整个区域分别进行PCR扩增,PCR产物测序后采用Blastn软件检索Genbank数据库,进行序列的同源性比较和分析。从所有识别度在99%-100%的序列中,选取来自不同菌纲、菌目和菌属的多个序列,用ClustalX软件对核苷酸序列进行多重序列比较,用MEGA 4.1软件作系统进化树分析。构建方法分别选择基于距离的邻近法(neighbor joining, NJ)和最大简约法(maximum parsimony, MP)。两种方法均选择了Bootstrap法进行进化树的验证分析。6整合子-耐药盒基因的克隆及克隆子的功能分析选择典型菌株,用PCR方法分别扩增位于可接合传递质粒上的典型1类整合子耐药基因盒基因pvl、位于基因组DNA上的2类整合子耐药基因盒基因pv2和整个2类整合子基因intI2pv2,分别将它们与克隆载体puc18连接,转化大肠杆菌DH5a,提取质粒酶切并进行特异PCR鉴定阳性克隆子。分别检测阳性克隆puc-pv1和puc-pv2对抗生素的最低抑菌浓度(minimum inhibition concentration, MIC)。分别用阳性克隆puc-intI2pv2和DH5a空菌作为受体菌,与供体菌志贺菌05100进行质粒接合传递实验,比较耐药性传递情况。7多重耐药志贺菌的无抗生素压力连续传代实验对耐药谱不同的、携带整合子不同的10株福氏志贺菌,进行无抗生素的多次传代培养。将所选菌株挑单个菌落经LB肉汤过夜增菌后,1：100倍LB肉汤稀释,继续37℃,200rpm振荡培养3-4h,至OD600值约为0.08-0.12之间后,继续1：100倍LB肉汤稀释37℃,200rpm振荡培养3-4h后,再次继续1：100倍LB肉汤稀释37℃,200rpm振荡过夜培养。如此1日3次,连续培养10日。在此期间,每隔5次传代就进行菌株的分离,并进行MIC的药敏实验和整合子的PCR检测。结果1志贺菌属耐药性和整合子分布的检测90株志贺菌(83株福氏志贺菌,7株宋内志贺菌)对TET、NAL、AMP、SXT和CHL的耐药率较高,分别为93.3%、92.2%、91.1%、82.2%和75.6%；多重耐药率达95.6%(86/90),最常见的耐药谱为AMP-TET-SXT-CHL-NAL(31/90,34.4%)。整合酶基因阳性率为87.8%(79/90),其中intI1单独阳性率3.3%；intI2单独阳性率10%；intI1和intI2同时阳性率为74.4%,3类整合子整合酶基因未检出。多重耐药菌的整合酶阳性率89.5%(77/86)。intI2阳性率在多重耐药株和非多重耐药株中分布差异有统计学意义(p<0.05)。2志贺菌属中整合子-耐药基因盒的特征分析共发现4种整合子耐药基因盒,序列提交Genbank获得登录号分别为FJ895301、FJ895302、GQ214137和EF634237。56株多重耐药志贺菌同时携带3种整合子-耐药基因盒系统：位于可接合传递质粒上的典型1类整合子耐药基因盒dfrA17-aadA5或dfrA12-orfF-adA2、位于染色体DNA上的非典型1类整合子耐药基因盒blaoxa-30-aadA1和位于染色体DNA上的2类整合子耐药基因盒dfrA1-sat1-aadA1非典型1类整合子耐药基因盒laoxa-3o-aadAl在对AMP-TET-CHL联合耐药菌中的阳性率高于非联合耐药菌,差异有统计学意义(p<0.05)3志贺菌属整合子-耐药基因盒的序列测定和系统发生分析序列分析和系统发生分析发现,除整合酶基因intll和非典型1类整合子耐药基因盒blaoxa-30-aadA1外,1类整合子耐药基因盒dfra17-aadA5、dfaA12-orf-aadA2以及2类整合子整合酶intI2基因、耐药基因盒dfrAl-satl-aadAl都存在基因分化现象。4志贺菌属整合子-耐药基因盒的克隆及克隆子功能分析1类整合子耐药基因盒dfrA17-aadA5阳性克隆使DH5a对STR的MIC从0.5μg/ml提高至4μg/ml,对TMP的MIC从2μg/ml提高至64μg/ml。2类整合子耐药基因盒dfrA1-Sat1-aadA1阳性克隆使DH5a对STR的MIC从0.5μghn1提高至32μg/ml,对TMP的MIC从2μg/ml提高至64pg/ml。通过质粒接合传递实验,来自志贺菌05100含有1类整合子耐药基因盒dfrA17-aadA5的质粒未对受体菌DH5a的抗生素敏感性产生影响,而来自志贺菌06208含有1类整合子耐药基因盒dfrA12-orf-aandA2的质粒使得受体菌DH5a获得对SXT和AMP的耐药性。对于相同供体菌福氏志贺菌05100而言,含有2类整合子克隆的大肠杆菌,与大肠杆菌空菌一样,经质粒接合传递后,多种抗生素的MIC并无变化。5多重耐药志贺菌的无抗生素压力连续传代实验无抗生素压力的连续30次传代实验发现：志贺菌05015、05105、06208分别丢失了对CHL、SXT和CIP的单一耐药性,其整合子-耐药基因盒系统无变化；志贺菌06218丢失了原有的全部耐药性(TXT.SXT和AMP),同时其仅有的位于基因组上的2类整合子-耐药基因盒系统,即intl2基因以及耐药基因盒dfrA1-Snt1-aadA1,也发生了丢失。结论1.志贺菌属细菌对常用抗生素普遍耐药,多重耐药现象严重(95.6%)。intl2基因阳性与志贺菌属多重耐药有关；非典型1类整合子耐药基因盒blaoxa-30-aadA1阳性与志贺菌属AMP-TET-CHL联合耐药有关。2.首次报道多重耐药志贺菌可同时携带3种不同整合子-耐药基因盒：位于可接合传递质粒上的典型1类整合子、位于染色体上的非典型1类整合子和2类整合子。3.来自不同菌属的整合酶基因和耐药基因盒在系统发生方面存在不同程度的分化。4.志贺菌属整合子携带的耐药基因盒仅针对特定抗生素产生特异性耐药,不同耐药基因盒致耐药程度不一。5.志贺菌属含整合子的质粒可接合传递与整合子无关的其他抗生素耐药性。6.志贺菌属2类整合子-耐药基因盒系统可能通过参与其他基因元件的移动和活动,来参与多个耐药基因结构的聚集或切除,从而与志贺菌属的多重耐药表型之间产生复杂的间接关联。更多还原

【Abstract】 As an important pathogen of infectious diarrhea, Shigella spp. can cause bacillary dysentery, also known as shigellosis. Shigellosis is the third high incidence in infectious diseases and the emphasis of prevention work in infectious diseases in China. Antibiotics have been used to cure shigellosis clinically for a long time. Antimicrobial therapy has been effective not only in allaying the dysenteric syndrome of shigellosis, but also reducing the duration of illness and the fecal excretion of the bacterium which preventing the further transmission. However, with the wide use of antibiotics, resistant isolates of Shigella spp., especially multiresistant isolates with multi-drug resistance (MDR), occurred. In case of shigellosis caused by a multidrug-resistant isolate, the safe and effective treatment options is limited both in developing and developed country. The multiresistance of Shigella spp. will cause not only clinical failures in the treatment of shigellosis but also the public health crises with persisting and spreading worldwide as a multidrug resistant organism. Therefore, the MDR of Shigella spp. and its molecular mechanisms have been paid great attentions.Antibiotic resistance of Shigella spp. can be attained through intrinsic or acquired mechanisms. Intrinsic mechanisms are those such as gene mutations found on chromosome, for example, mutations within the drug’s targets, DNA gyrase and topoisomerase, to get the resistance to quinolones, and those such as enzymatic mechanisms of drug modification, altered membrane permeability and enhanced efflux pump expression, to get MDR. Acquired mechanisms involve the extrachromosomal elements acquired from other bacteria in the environment. Although classically attributed to chromosomal mutations, resistance is most commonly associated with acquired mechanisms. There are different types of mobile DNA segments such as plasmids, transposons, and bacteriophages which can transfer resistance determinants among organisms within the same genus or between distant organisms as gram-positive and gram-negative bacteria and then contribute to multidrug resistance in bacteria. Recently, integrons have been observed to take part in the transfer of resistance genes. Integrons are genetic elements composed of an integrase, gene cassettes and the site-specific recombination site for gene cassettes. Integrons themselves are not mobile but integrase can excise and integrate the gene cassettes from and into the integron. Integrons can reside on transposons, conjugative plasmids, or the chromosome, facilitating the acquisition and dissemination of gene cassettes and so accounting for their wide distribution and their significant association with the multiresistance phenotype in Enterobacteriaceae. Many studies showed that integron containing isolates were more likely to be resistant to drugs and integron-gene cassettes were related to the multiresistance of Shigella spp.. Class 2 integron was found more frequently than class 1 integron in Shigella spp. in studies all over the world. The types of gene cassettes discovered in isolates of Shigella were not various relatively. Neither the classes of integron nor the types of integrated cassettes were correlated completely to the resistant phenotype of Shigella spp.. The resistant phenotype could only be partially explained by the presence of integrons. It’s necessary to investigate the relationship among integron, gene cassettes and multiresistance, and to evaluate the status and contribution of integrons to the multiresistance of Shigella spp..In current study,83 isolates of S. flexneri and 7 isolates of S. sonnei were determined the susceptibility to antimicrobial agents and screened for the presence of integrons and gene cassettes. The clone and functional analysis of integrons and gene cassettes were conducted to investigate the contribution of integrons to the multiresistance of Shigella spp..Methods1 Susceptibility testNinety isolates of Shigella spp. were determined their susceptibility to eight antimicrobial agents, which were ampicillin (AMP), tetracycline (TET), trimethoprim-sulfamethoxazole (SXT), chloramphenicol (CHL), nalidixic acid (NAL), ciprofloxacin (CIP), gentamicin (GEN) and cefazolin (CFZ), by the disk diffusion method on Mueller-Hinton agar according to the recommendations of the Clinical and Laboratory Standards Institute. The minimal inhibitory concentration of TET, AMP, CHL, CIP, streptomycin (STR), trimethoprim (TMP) and sulfamethoxazole (SSS) were also conducted. Escherichia coli ATCC 25922 was used as a quality control strain.2 Screening for integrase and various region of integron by PCR and PCR-RFLPIntegrase of class 1, class 2 and class 3 were detected by PCR from the total DNA extracted by boiling method. Total DNA and plasmid DNA were extracted from all isolates respectively by Axyprep Bacterial Genomic DNA Miniprep Kit to detect the various regions of integrons. The primers hep58 and hep59 were designed to amplify the various region of typical class 1 integron, primers hep58 and ISR for that of atypical class 1 integron, and primers hep74 and hep51 for that of class 2 integron. The PCR products of various regions with similar length were examined by restriction fragment length polymorphism analysis with PvuⅠ, HindⅢor HinfⅠThe identical restriction profiles were regarded as the same array of gene cassettes.3 Southern blottingSouthern blotting analysis was applied according to the manufacturer’s instructions of DIG DNA labeling and detection kit. After electrophoresis of the total DNA digested by PstⅠand BamHⅠ, or the plasmid DNA in 0.8% agar gel, the DNA transferred to Hybond N+nylon membrane and fixed in hot air drying oven, the digoxigenin-labelled DNA probes specific to intI1 and intI2 by random primering were used to identify the location of integrons.4 Conjugation testConjugation was carried out by using E. coli DH5a as a recipient in all the 90 isolates. The over night culture of purified donor and recipient cells were diluted 100-fold in 2mL of fresh LB broth. The growth continued at 37℃until OD600 reached about 0.6. Then 0.1 mL each of the donor and recipient cultures was mixed in 1 mL of fresh LB broth. After incubation at 37℃without shaking for 16-18h, the mixture was plated on MacConkey agar. The morphological differences between E. coli and Shigella on MacConkey were used to select the recipients. The change of the susceptibility to antimicrobial agents was measured by the disk diffusion method as described above.5 Sequence analysis and phylogenetic tree of integron and gene cassettesThe integrase and various region of typical class 1 integron, atypical class 1 integron and class 2 integron were amplified by PCR respectively. The PCR products of representative isolates were sequenced and then analyzed by using software BLAST on the website of the National Center for Biotechnology Information. Several sequences from different class, order or genus were selected among all sequences with 99%-100% identify. Alignment of multiple sequences was conducted by the software ClustalX. The construction of phylogenetic tree was carried out by the neighbor joining (NJ) method and maximum parsimony (MP) method respectively using software MEGA 4.1. Bootstrap test was selected to validate the phylogeny in each process.6 Cloning and functional analysis of integron and gene cassettesThe interested PCR products (including the gene cassettes of typical class 1 integron located in conjugatable plasmid named as pvl, the gene cassettes of class 2 integron located in genome named as pv2, and the whole gene of class 2 integron named as intI2pv2) of representative isolates were cloned into pucl8 or pMD-18 by the Takara TA cloning kit, and then transformed into E. coli DH5a. The minimal inhibitory concentration to antibiotics of positive clones DH5a with puc-pvl and puc-pv2 were determined respectively. Conjugation was carried by using E. coli DH5a and positive clone DH5a with puc-intI2pv2 as recipient respectively, and S. flexneri 05100 as donor. The resistances to antibiotics of conjugants were compared.7 Continuous cultivation without antibiotic pressure for the multiresistant isolates of Shigella spp.Ten isolates of S. flexneri with different resistant phenotypes and positive integrons were cultured continuously without any antibiotic pressure. Single clone of each isolate was inoculated in fresh LB broth at 37℃overnight. On day one, the suspension was diluted 100 times in fresh LB broth and cultured continuously at 37℃with 200rpm shaking for 3-4h till OD600 reaching about 0.08-0.12. The process was repeated three times a day. The same process was repeated for each isolate for 10 days. The isolate after every 5 inoculation was subjected to the suscepbility test and detection of integrons by PCR.Results1 Prevalence of resistance and integrons in Shigella spp.Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) showed high resistant rate to TET, NAL, AMP, SXT and CHL at 93.3%,92.2%,91.1%,82.2%and 75.6% respectively. Multidrug resistance was detected in 86 isolates (95.6%). Among all the Shigella spp., the most frequent resistant profiles were AMP-TET-SXT-CHL-NAL (31/90,34.4%). PCR analysis revealed that 79 (87.8%) isolates carried integron of class 1 only (3.3%), class 2 only (10.0%) and both (74.4%). No intI3 was found.89.5%(77/86) isolates of multidrug resistant Shigella spp. carried integron. The prevalence of intI2 was significantly different between multidrug resistant isolates and non-multidrug resistant isolates (p<0.05).2 Characteristics of integron and gene cassettes in Shigella spp.Four gene cassette arrays were found. The sequences were submitted to the GenBank database and the accession numbers are FJ895301, FJ895302, GQ214137 and EF634237 respectively. There were overall 56 isolates of multiresistant S. flexneri carrying three integrons simultaneously. They are the gene cassettes of classic class 1 integron dfrA17-aadA5 or dfrA12-orfF-adA2 located in conjugatable plasmid, the gene cassettes of atypical class 1 integron blaoxa-30-aadA1 located in genome and the gene cassettes of class 2 integron dfrA1-sat1-aadA1 located in genome.The prevalence of gene cassettes of atypical class 1 integron blaoxa-30-aadA1 in isolates resistant to AMP-TET-CHL association was significant higher that in isolates without resistance to AMP-TET-CHL association (p<0.05).3 Sequence analysis and phylogenetic tree of integron and gene cassettesExcept for integrase gene of class 1 integron intI1 and gene cassettes blaoxa-30-aadA1 of atypical class 1 integron, the sequences of following fragments, gene cassettes of class 1 integron dfrA17-aadA5, dfrA12-orf-aadA2, integrase gene of class 2 integron intI2, and gene cassettes of class 2 integron dfrA1-sat1-aadA1, were all found having genetic differentiation.4 Cloning and functional analysis of integron and gene cassettesThe positive clone of DH5a with puc-pv1 (dfrA17-aadA5) increased the MIC to STR from 0.5μg/ml to 4μg/ml, the MIC to TMP from 2μg/ml to 64μg/ml. The positive clone of DH5a with puc-pv2 (dfrA1-sat1-aadA1) increased the MIC to STR from 0.5μg/ml to 32μg/ml, the MIC to TMP from 2μg/ml to 64μg/ml.No resistance was transferred by conjugation from the plasmid of S. flexneri 05100 carrying gene cassettes of class 1 integron dfrA17-aadA5 to the recipient DH5a. However, the resistance to SXT and AMP was transferred by conjugation from the plasmid of S. flexneri 06208 carrying gene cassettes of class 1 integron dfrA12-orf-aadA2 to the recipient DH5a. With S. flexneri 05100 as the same donor, no changes of MIC to antibiotics were detected after conjugation with the positive clone of DH5a with puc-pv2 (dfrA1-sat1-aadA1) and DH5a as recipient respectively.5 The results of continuous cultivation without antibiotic pressure for the multiresistant isolates of Shigella spp.After 30 times of inoculation without antibiotic pressure, the isolate of S. flexneri 05015,05105,06208 lost the single resistance to CHL, SXT and CIP respectively without any change of the integrons’status. The isolate of S. flexneri 06218 lost all the resistance (to TXT, SXT and AMP) as well as the only integron, class 2 integron and gene cassettes dfrA1-sat1-aadA1, located in genomic DNA.Conclusions1. The isolates of Shigella spp. are usually resistant to common antibiotics and most of them are multidrug resistant (95.6%). The positive of intI2 is related to the multiresistance of Shigella spp. The positive of gene cassettes blaoxa-30-aadA1 of atypical class 1 integron is related to the associated resistance to AMP-TET-CHL.2. It’s the first report of coexistence of three integons and gene cassettes, the classic class 1 integron located in conjugatable plasmid, the atypical class 1 integron located in genome and the class 2 integron located in genome.3. The genetic differentiations occur among sequences of integrase and gene cassettes from different class, order or genus.4. The gene cassettes of integron in Shigella spp. lead to the specific resistance and the level of resistance was different in different gene cassette.5. The plasmid carrying integron in Shigella spp. can transfer the resistance unrelated to the integron.6. The class 2 integron and resistant gene cassettes may take part in the assemble or excise of multiple resistant genetic structure by participating the activity of other mobile DNA segments, accordingly having indirect association with the multiresistance of Shigella spp..更多还原