【作者】 苑丽；

【导师】 胡功政；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2010， 博士

【摘要】 随着抗菌药长期广泛使用,肠杆菌科鸡源分离菌对药物的耐药率逐年升高,且呈多重耐药趋势。导致细菌获得多重耐药的因素有很多,其中超广谱β-内酰胺酶(extended spectrumβ- lactamases,ESBLs)和整合子被证实常参与细菌多重耐药的形成。为此,做了以下研究:(一)利用VITEK-32全自动细菌鉴定仪对从河南省14个地市不同养鸡场分离的64株鸡源分离菌进行鉴定,获得51株鸡源大肠杆菌、8株鸡源沙门菌、4株鸡源奇异变形杆菌和1株鸡源鲍曼不动杆菌,其中鸡源鲍曼不动杆菌为国内外首次分离。采用微量稀释法测定64株鸡分离菌对β-内酰胺类、氨基糖苷类、四环素类、氯霉素类、氟喹诺酮类和磺胺等六类共24个药物或组合的多重耐药表型。结果发现58.8%~74.5%的鸡源大肠杆菌对三代头孢耐药,96%(49/51)为四重及以上耐药菌株,92.2%(47/51)呈五重及以上耐药,82.4%(42/51)呈六重及以上耐药。8株鸡源沙门菌的多重耐药谱均不低于四重,且其中两株菌呈现出同时耐12重药物。其他五株分离菌的耐药谱均低于3重(其中Dpro为五重耐药除外),且均对三代头孢敏感。(二)ESBL检测结果表明54.7%(35/64)受试菌为产ESBL菌。利用PCR法检测64株受试菌携带β-内酰胺酶基因情况,结果发现38株携带TEM基因,包括TEM-1亚型37株,TEM-57亚型1株,其中TEM-57亚型为首次在鸡源分离菌中检出。19株携带CTX-M基因,包括14株CTX-M-65,1株CTX-M-14,3株CTX-M-24和1株CTX-M-90,其中CTX-M-90为首次发现并命名的一个新基因亚型,CTX-M-65亚型为首次在动物源分离菌中检出。由此得出CTX-M基因突变过程为CTX-M-14→CTX-M-24或CTX-M-90→CTX-M-65。21株携带OXA基因,包括11株OXA-1,2株OXA-2和16株OXA-10。说明TEM-1是目前河南省养鸡场鸡源分离菌中的主要基因亚型,其次为OXA-10、CTX-M-65和OXA-1。(三)分析产ESBL菌和非产ESBL菌携带β-内酰胺酶基因情况和多重耐药谱。结果发现74.3%产ESBL菌(26/35)同时携带两种以上β-内酰胺酶基因,而仅6.9% (2/29)非产ESBL菌同时检出2种β-内酰胺酶基因。在35株产ESBL菌中,100%耐氨苄西林,对三代头孢的耐药率为77.1%~91.4%,对三代头孢/酶抑制剂联用的耐药率为0~37.1%,同时,产ESBL菌的多重耐药谱均不低于六重,其中94.3%(33/35)为七重及以上耐药菌株。而非产ESBL菌对β-内酰胺类的耐药率均低于45%(氨苄西林除外),44.8% (13/29)的多重耐药谱不低于6重,仅20.7%(6/29)不低于七重。比较产酶菌ESBL基因型与多重耐药谱关系后发现47.4%(9/19)产CTX-M-ESBL菌多重耐药谱不低于十重,而在16株非产CTX-M-ESBL菌中,仅18.8%(3/16)不低于十重。同时还发现在15株产CTX-M-65或CTX-M-90菌株中,60%(9/15)多重耐药谱超过10重,20%(3/15)为12重;而4株携带CTX-M-14或CTX-M-24菌株多重耐药谱均低于10重。即携带CTX-M-65或CTX-M-90的菌株对受试药物耐药性较携带CTX-M-14或CTX-M-24菌株严重,提示CTX-M基因亚型的突变趋势与细菌耐药表型扩大延伸方向基本一致。(四)采用1/2 MIC头孢曲松或头孢噻肟诱导培养5株非产ESBL鸡源大肠杆菌和O78。结果发现培养至10代时,头孢曲松或头孢噻肟的MIC值升高了8~64倍;培养至20代时,两药的MIC值又升高了2~16倍,其中头孢噻肟诱导的O78已对头孢噻肟耐药;培养至30代时,诱导菌对头孢曲松、头孢噻肟、庆大霉素、阿米卡星、多西环素、氟苯尼考和恩诺沙星均耐药。说明在单一抗菌药定向选择性压力下,细菌表达多重耐药表型的速度是较快的。(五)经PCR检测及测序分析,未诱导前O78为TEM-1型,头孢噻肟诱导至20代时,TEM序列发生一处碱基变异(24T→C);诱导至30代时,TEM-1序列发生两处碱基变异(24T→C,40C→T),其中后一处碱基突变引起14Pro→Ser,即TEM-1基因可直接突变为一个新基因亚型,说明药物定向诱导对菌株TEM基因变异起了重要作用。未诱导前,受试菌均不携带CTX-M基因,诱导至20代时,头孢噻肟诱导O78已突变为CTX-M-14-ESBL菌株;诱导至30代时,所有诱导菌株均成为CTX-M-ESBL(CTX-M-90和CTX-M-65)菌株,说明在三代头孢定向选择性压力下,CTX-M型基因具有较快变异速度。分析各CTX-M基因序列推断出CTX-M基因突变过程:非产ESBL菌首先获得CTX-M-14基因,接着该基因发生氨基酸点突变成为CTX-M-90,然后CTX-M-90基因进一步积累氨基酸点突变,菌株获得CTX-M-65基因,随着诱导的深入,CTX-M-65基因也开始出现碱基突变,但由于该突变仅为沉默突变,故暂时未有新基因亚型产生。此外,本诱导试验获得的CTX-M基因亚型均在前期鸡源分离菌检测时有检出,说明本诱导试验的推断出的CTX-M基因突变过程可在一定程度上反映出临床菌实际的突变过程,诱导药物与临床菌实际接触的药物相似。(六)利用PCR技术检测51株鸡源大肠杆菌中整合子流行情况。结果表明86.3%(44/51)检出Ι类整合子,3.9%(2/51)检出Ⅱ类整合子,未检出Ⅲ类整合子和非典型Ι类整合子。基因盒插入区片段经克隆、测序和比对后发现在44株Ι类整合子中耐药基因盒组合类型包括sat(100%)、dfrA1/aadA1(45.4%)、dfrA17/aadA5(22.5%)、dfrA1/sat/aadA2(6.8%)和约4800 bp的未知基因盒(27.3%),其中dfrA1/sat/aadA2基因组合为国内外首次报道。进一步采用巢式PCR和PCR定位技术检出4800 bp未知基因盒内含有blaCTX-M基因,且该基因位于基因盒区的中下游。两株Ⅱ类整合子阳性菌中检出的耐药基因盒组合均为dfrA1/sat1/aadA1。(七)分析鸡源大肠杆菌整合子与ESBL的关系后发现93.6%产ESBL鸡源大肠杆菌(29/31)携带整合子,而在20株非产ESBL鸡源大肠杆菌中,此数值为75%,两组之间的差异具有统计学意义,说明整合子在产ESBL鸡源大肠杆菌中分布更为集中。在15株携带整合子的非产ESBL菌中,80%菌株(12/15)携带一种整合子,且整合子的可变区仅整合了一种耐药基因;而对29株携带整合子的产ESBL菌来说,有89.6%菌株(26/29)携带2~4种整合子。更多还原

【Abstract】 ABSTRACT With the widespread use of antimicrobial agents for prophylaxis and therapy of infected animals, the wild dissemination of antimicrobial resistance, especially multidrug-resistance (MDR), is increasing among chicken Enterobacteriaceae in the worldwide. There are many ways for bacteria to obtain multidrug-resistance, example for extended-spectrumβ-lactamases (ESBLs) and integrons. Therefore, the present study was delineated to identify and characterize.1. Sixty-four chicken Enterobacteriaceae isolates were identified with the Vitek-32 automatic microbe system, which were isolated from different chicken farms, Henan province. All isolates were as follows: Escherichia coli (n=51), Salmonella (n=8), Proteus mirabilis (n=4), and Acinetobacter baumannii (n=1). To our knowledge, Acinetobacter baumannii was isolated firstly from chickens. The MDR profiles of 64 clinical isolates were determined at the same time using the CLSI microbroth dilution method. The antimicrobial agents containedβ-lactam antibiotics, aminoglycosides, tetracyclines, amphenicols, fluoroquinolones, and sulfanilamide. The results demonstrated that 58.8%~74.5% E.coli showed resistance to third generation cephalosporins, 96% (49/51) were resistant to more than four antimicrobial agents, 92.2% were more than five, and 82.4% were more than six. The MDR profiles of 8 Salmonella isolates were more than four. The other isolates were sensitive to third generation cephalosporins, and their MDR profiles were not more than three.2. The ESBLs detection showed that 54.7% (35/64) isolates were ESBL-producing bacteria. With the PCR, BlaTEM was detected in 38 isolates, blaCTX-M was detected in 19 isolates and 21 isolates harboured blaOXA among 64 strains. Except for one isolate that had TEM-57, which was first detected in chicken E.coli, most of blaTEM isolates possessed TEM-1. The blaCTX-M isolates encoding CTX-M-65, CTX-M-14, CTX-M-24, and CTX-M-90 were 14, 1, 3, and 1 among 19 CTX-M-ESBL isolates, respectively. The CTX-M-90 gene was the new subtype in all ESBL genotypes. Based on the amino acid sequences of four CTX-M subgenes, we postulated possible evolution scheme was CTX-M-14→CTX-M-24 or CTX-M-90→CTX-M-65. The blaOXA contained OXA-1 (11 strains), OXA-2 (2 strains), and OXA-10 (16 strains). These findings indicated that TEM-1 was the most frequently encountered blaTEM allele in chicken clinical bacterial populations, Henan. Then the more commonβ-lactamase alleles were OXA-10, CTX-M-65, and OXA-1.3. Twenty-six out of 35 ESBL-producing isolates (74.3%) carried more than two bla genes, whereas, only 6.9% (2/29) non-ESBL-producing isolates harboured two bla genes. The resistant rates of 35 ESBL-producing isolates to ampicillin, third generation cephalosporins, and third generation cephalosporins/beta-lactamase inhibitors were 100%, 77.1%~91.4%, and 0~37.1%. The MDR profiles of those isolates were more than six, and 94.3% (33/35) were more than seven. But, the resistant rates of 29 non-ESBL-producing isolates toβ-lactam antibiotics were lower than 45% (except ampicillin), only 44.8% (13/29) owned more than six MDR profiles, and 20.7%(6/29)were more than seven. The MDR profiles of 47.4% CTX-M-ESBL isolates (9/19) were not less than ten, yet that was only 18.8% (3/16) among non-CTX-M-ESBL isolates. At the same time, the results showed that 60% isolates harbouring blaCTX-M-65 or blaCTX-M-90 had more than ten MDR profiles, 20% (3/15) had more than twelve, but the other blaCTX-M isolates demonstrated less than ten. The resulte suggested that the CTX-M-65 and CTX-M-90 subgroups were more prone to exhibit the MDR phenotypes than the -14 and CTX-M-24, indicating that there has identity of the blaCTX-M evolutionary tendency and the MDR phenotypes extended trend.4. Five non-ESBL chicken E.coli and O78 strains were cultivating in broth, which contained ceftriaxone or cefotaxime with 1/2MIC subinhibitory concentrations. When the strains were cultivated to the tenth generation, the MICs of two drugs increased 8~64 times. The MICs increased 8~64 times again and the O78 strain induced by cefotaxime expressed resistance to cefotaxime when cultivated to the twentieth generation. The strains passaged to the thirtieth generation demonstrated resistance to ceftriaxone, cefotaxime, gentamicins, amikacin, doxycycline, florfenicol, and enrofloxacin, indicating that the speed of bacteria obtaining MDR genes and presenting MDR phenotypes was fast with antimicrobial agents selective pressures.5. With PCR and sequencing analysis, O78 strain harboured TEM-1 When the strain was induced to the 20th passage with cefotaxime, the nucleotide sequence occurred one base mutation (24T→C). It had two base mutations (24T→C, 40C→T) at the 30th generation, and the latter mutation led to a new amino acid substitution (14Pro→Ser), that is, TEM-1 subtype evolved to a new subtype. This result illustrated that the antimicrobial agents selective pressures play an important role in blaTEM evolations. All strains did not carry any blaCTX-M before inductions. The O78 strain had changed to a CTX-M-14- ESBL strain When it was cultured to the twentieth generation with cefotaxime. At the thirtieth generation, All the strains had evolved to CTX-M-ESBL strains (CTX-M-90 or CTX-M-65). This finding demonstrated that the variable speed of blaCTX-M was higher than that of blaTEM. We could mainly concluded the evolutionary process of blaCTX-M by analysis the different CTX-M amino acid sequences. With the drugs selective pressures, the non-ESBL strains firstly obtained blaCTX-M-14, then blaCTX-M-90, and then blaCTX-M-65. It is worth noting that CTX-M subtypes obtained from inductive trials are detected simultaneously in chicken isolates, which suggested that the blaCTX-M evolutionary process could partially reflect that of isolates.6. Forty-four of the 51 chicken E.coli (86.3%) detected class 1 integron and 3.9% (2/51) contained class 2 integron. Five gene arrangements have been detected among class 1 integrons, containing sat (100%), dfrA1/aadA1 (45.4%), dfrA17/aadA5 (22.5%), dfrA1/sat/aadA2 (6.8%), and 4800 bp unknown. cassette array. To our knowledge, dfrA1/sat/aadA2 was a novel gene cassette array. blaCTX-M was detected in 4800 bp unknown cassette array and located the downstream by nested PCR and PCR mapping. Class 2 integrons contained the one array gene cassette of dfrA1/sat1/aadA1.7. The results showed that 93.6% ESBL-producing chicken E.coli (29/31) carried integrons, but the value was 75% among non-ESBL strains. The difference between two groups has a statistical significance, which illustrated that the ESBL-producing E.coli are more prone to harbour integrons. Among 15 non-ESBL strains with integrons, 90% isolates (12/15) carried only an integron, and the variable region only integrated one resistant gene cassette. But 89.6% ESBL strains with integrons (26/29) contained 2~4 integrons.更多还原