【作者】 潘玉善；

【导师】 胡功政；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2012， 博士

【摘要】 随着抗菌药物的广泛使用,禽源肠杆菌科细菌对药物的抗性快速传播,尤其呈多重耐药的细菌更为明显。过去十几年,产超广谱β-内酰胺酶的大肠杆菌已经广泛流行,并呈现多样性,这很多程度上是由于CTX-M型超广谱β-内酰胺酶基因的出现。该型基因常常在人类、食品动物、伴侣动物、野生动物和环境中发现。有多种原因使细菌能获得多重耐药,如接合转移、转座、插入序列元件、编码位点特异性整合的DNA移动元件(整合子)。本试验的目的是研究禽源大肠杆菌、奇异变形杆菌多重耐药的分子特征及CTX-M型超广谱β-内酰胺酶基因传播扩散机制。通过药敏试验、ESBL和AmpC产酶确证、耐药基因检测、质粒分析、接合试验、质粒不相容性分群,研究禽源细菌的多重耐药特性；通过系统发育分群和多位点序列分型研究禽源大肠杆菌的进化关系。通过PCR定位技术和分子克隆技术分析bluCTX-M的基因环境和rmtB基因的基因环境。1.利用VITEK-32全自动细菌鉴定仪对从不同地区养殖场的病死家禽中分离的64株禽源分离菌进行鉴定,获得43株禽源大肠杆菌和21株奇异变形杆菌。ESBL和AmpC的确证试验结果表明：43株禽源大肠杆菌中有19株产超广谱β-内胺酰酶,1株产AmpC酶；分离的21株奇异变形杆菌中,有10株产超广谱β-内胺酰酶,6株产AmpC酶。药物敏感性试验表明超广谱β-内胺酰酶的禽源大肠杆菌和奇异变形杆菌表现不同的耐药特征。大部分菌株对氨苄西林、头孢曲松、头孢噻肟、卡那霉素耐药。此外,有4株大肠杆菌对氨基糖苷类药物(卡那霉素、庆大霉素、阿米卡星)呈现高水平耐药。本试验系统发育分析表明：4株大肠杆菌属于D群,其余属于A群。多位点序列分型(Multilocus sequence typing,MLST)结果表明：20株大肠杆菌属于11种ST序列,表现了高的差异性。2.本试验通过接合试验获得了15株大肠杆菌的接合子。大肠杆菌耐药基因检测表明：16株大肠杆菌产TEM-1型β-内酰胺酶；19株产CTX型超广谱β内酰胺酶,其中1株为CTX-M-3,5株为CTX-M-14,2株为CTX-M-55,1株为CTX-M-24,1株为CTX-M-27,9株为CTX-M-65。5株产OXA-1型超广谱β-内酰胺酶：1株产CMY-2型AmpC酶；奇异变形杆菌耐药基因检测表明：10株产TEM型β-内酰胺酶；10株产CTX型超广谱β-内酰胺酶,占48%(10/21),其中CTX-M-14型的6株,CTX-M-65型的4株；6株产CMY-2型AmpC酶,占29%(6/21)；6株产OXA-1型超广谱p-内酰胺酶,其中的1株还同时产OXA-10超广谱β-内酰胺酶；SHV型基因在两种菌中均没有检测到。3.blaCTX-M的基因环境因其亚型不同而有差异。blaCTX-M-3、blaCTX-M-55、 blaCTX-M-14、blaCTX-M-24、blaCTX-M-27、blaCTX-M-65上游均存在ISEcp1,但某些情况被IS26截断或被IS10插入。重要的是,研究首次确定了一种新的blaCTX-M基因环境的结构ISEcp1Δ-IS1294-ΔISEcp1-blaCTX-M-5-ORF477,在此结构中,ISEcp1被一个完整的IS1294截断,已提交NCBI获得序列号JN977127。上游ISEcp1与blaCTX-M基因的间隔区也有差异,对blaCTX-M-55是45bp,也有48bp,对CTX-M-9族基因均为42bp。对于下游,核酸序列分析表明,CTX-M-1族的blaCTX-M-3和blaCTX-M-55是orf477,对CTX-M-9族是IS903,但有的被截断。结果也同时表明blaCTX-M基因多位于IncI1和IncF可接合性质粒上,blacmy-2基因位于IncA/C可接合性质粒上。4.4株大肠杆菌中检测到rmtB基因(9%,4/43),占产β-内酰胺酶基因的大肠杆菌20%(4/20)。值得注意的是,含有这种基因的大肠杆菌还同时产CTX-M型ESBL和CMY-2型AmpC。这对临床使用β-内酰胺类抗生素和氨基糖苷类抗生素构成了严重威胁。在1株大肠杆菌中,发现blaCTX-M-55和rmtB基因分别位于不同的质粒上IncI1和IncN,这种抗性质粒可以单独也可以一起通过接合而传递,结果也同时显示rmtB基因被Tn3转座子携带。综上,产ESBL的大肠杆菌在禽类中广泛流行,CTX-M也呈多样性流行。blaCTX-M的基因环境因亚型不同而有差异。核酸序列表明,blaCTX-M基因的上游存在ISEcp1元件;blaCTX-M基因的下游在CTX-M-1组是orf477,在CTX-M-9族是IS903。本试验中,在大肠杆菌C21中同时检测到blaCTX-M-55和rmtB基因,并且分别位于不同的接合型质粒上。同时,第一次检测到ISEcp1Δ-IS1294-ΔISEcp1-blaCTX-M-55-ORF477的结构,ISEcp1被一个完整的IS1294截断。质粒接合传递、转座和IS元件有助于多重耐药基因的快速传播,这对人类和兽医构成了很大的威胁。更多还原

【Abstract】 With the widespread use of antimicrobial agents for prevention and therapy in infected animals, the rapid dissemination of antimicrobial resistance, especially multidrug-resistance (MDR), is increasing among poultry-original Enterobacteriaceae in the worldwide. Extended-Spectrum-β-Lactamase (ESBLs)-producing E. coli have increased in prevalence and diversity through the last decade, largely due to the emergence of CTX-M beta-lactamases. CTX-M producing E. coli have been found in humans, food producing animals, companion animals, wild animals and the environment. There are many ways for bacteria to obtain multidrug-resistance, example for Conjugative transfer, transposition,IS elements and mobile DNA elements encoding site-specific gene-integration functions (integrons). The purpose of this study was to characterize the molecular characterisation and mechanism of dissemination of CTX-M genes in Multidrug-resistant isolates of Escherichiu coli (E. coli) and Proteus mirabilis(p. mirabilis) from Poultry.The Multidrug-resistant isolates were examined by isolation and identification, antimicrobial susceptibility testing, confirmation of ESBL and AmpC, resistant genes screening, plasmid analysis, conjugation experiments, and the plasmid incompatibility groups in parental strain and its transconjugants was investigated by PCR-based replicon typing. The multiplex PCR was carried to determine whether this strain were commensal (A and B1) or were associated with phylogroups exhibiting extraintestinal virulence (B2and D). Multilocus sequence typing (MLST) was used to study the genetic relatedness of a collection of isolates of E. coli. PCR mapping and gene cloning were performed to investigate the genetic environment of blaCTX-M and rmtB, respectively.1. Forty-three isolates of E. coli and twenty-one isolates of p. mirabilis were identified with Vitek-32system (BioMerieux, France), which were collected from the dead poultry from different geographic regions. The results of ESBL and AmpC confirmation showed that19isolates of E. coli produced the ESBLs,1isolate of E. coli produced the AmpCs,10isolates of p. mirabilis produced the ESBLs,6isolate of p. mirabilis produced the AmpCs. The ESBL-producing isolates of E. coli and the isolates of p. mirabilis showed different patterns of antibiotic resistance based on their resistant phenotype by antimicrobial susceptibility testing. Most of these isolates were resistant to ampicillin, ceftriaxone, cefotaxime, kanamycin. Moreover,4isolates of E. coli also exhibited a high level resistance to aminoglycosides including kanamycin, amikacin, gentamicin. In the present study, Phylogenetic analysis indicated that the4isolates of E. coli belonged to group D, the other belonged to group A. The results of MLST revealed that20isolates of E. coli belonged to11kinds(ST23, ST93, ST117, ST155, ST156, ST162, ST224, ST410, ST539, ST602, ST2847). MLST analysis showed a high diversity of the isolates.2.15transconjugants from20E. coli isolates were successfully obtained by conjugation experiments.BlaTEM-1genes were detected in16of the43E. coli isolates (37%). BlaTEM-M genes were detected in19of the43E. coli isolates (44%). The most common CTX-M types were blaCTX-M-65(n=9),blaCTX-M-14(n=5), followed by blaCTX-M-55,blaCTX-M-3,blaCTX-M-24,blaCTX-M-27.BlaOXA-1was detected in5isolates. blacny-2was detected in one isolate.qnrS1gene was detected in1of the43E. coli isolates (2.3%). BlaTEM-1genes were detected in10of the21p. mirabilis isolates (48%). blaCTX-M genes were detected in10of the21p. mirabilis isolates (48%). The most common CTX-M types were blaCTX-M-14(n=6), blaCTX-M-65(n=4). blacmy-2was detected in6isolates (29%). qnrD gene was detected in3of the21E. coli isolates (14%). BlaOXA-1was detected in6isolates, and BlaOXA-10was detected one same isolate simultaneously. BlaSHV was not detected in all isolates.3. The genetic environments of blaCTX-M genes were shown specific for the subtype of the genes.A mobile genetic element ISEcpl (in some cases truncated or inserted by IS26, IS10sequences) were detected upstream of blaCTX-M-3, blaCTX-M-55, and blaCRX-M-14,blaCTX-M-24, blaCTX-M-27, blaCTX-M-65genes. Importantly, for the first time, a new arrangement, ISEcp1Δ-IS1294-ΔISEcpl-blaCTX-M-55-ORF477was identified, in which the ISEcpl element was disrupted by another insertion sequence, IS1294. The nucleotide sequence of the genetic environment of blaCTX-M-55was deposited in GenBank under the accession no. JN977127. A special characteristic was the sequence between ISEcp1and blaCTX-M gene:48bp for blaCTX-M-55and45bp for blCTX-M-55;42bp for CTX-M-9group genes. Nucleotide sequence analysis revealed that orf477was located downstream of blaCTX-M-3and blaCTX-M-55, and insertion sequence IS903(intact or truncated) was detected downstream of CTX-M-9group gene. It was also shown that blaCTX-M genes were mostly located on conjugative plasmids belonging mainly to the incompatibility groups IncI1and IncF, while conjugative plasmids, which harboring blacmy-2, belonged IncA/C.4. The rmtB gene was detected in4of the43E. coli isolates (9%), in4of the20beta-lactamase-producing E. coli isolates(20%). It is worth noting that CTX-M-producing isolates of E. coli coproduced16S rRNA methylase, and cmy-2-producing isolate of E. coli coproduced16S rRNA methylase, and this poses a severe threat to the clinical use of β-lactam antibiotics and aminoglycosides in human and veterinary medicine. The blaCTX-M-55and rmtB genes were found to be present in the separate plasmids belong to the IncI1and IncN, respectively. These antibiotic resistant plasmids could be transferred to the recipient strain alone or together. The results also showed that the rmtB gene was carried by Tn3transposonIn conclusion, CTX-M-producing E. coli have increased in prevalence and diversity in poultry. The genetic environments of blacCTX-M genes were shown specific for the subtype of the genes.Nucleotide sequence analysis revealed that a mobile genetic element ISEcp1were detected upstream of blaCTX-M, insertion sequence IS903(intact or truncated) was detected downstream of CTX-M-9group gene, and orf477was located downstream of CTX-M-1group gene. The blaCTX-M-55and rmtB genes were detected firstly in the same strain of E. coli C21isolated from a chicken in China, and were found to be present in separate plasmids belonging to the Incl1and IncN families, respectively.Both plasmids harboring these genes were conjugative. This study also described, for the first time, a new arrangement,ISEcp1Δ-IS1294-AISEcp1-blaCTX-M-55-ORF477, in which the ISEcpl element was disrupted by another insertion sequence, the IS1294element. Conjugative plasmid, transposition and IS elements may contribute to the rapid dissemination of antibiotic resistance genes, and this is of great significance to human and veterinary medicine.更多还原