【作者】 张春辉；

【导师】 胡功政；

【作者基本信息】 河南农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 为克服细菌因产生β-内酰胺酶而对β-内酰胺环类抗生素耐药的问题，本文进行了猪鸡常见致病菌β-内酰胺酶的鉴定检测，并对β-内酰胺酶抑制剂舒巴坦的抑酶保护作用，进行了初步研究。首先用试管二倍稀释法，测定了常见β-内酰胺环类抗生素对猪鸡常见致病菌的体外抗菌活性；进一步选择对氨苄西林、阿莫西林严重耐药的大肠杆菌分离株用碘测定法、酸测定法、和Nitrocefin纸片法进行了β-内酰胺酶的鉴定检测；同时用试管二倍稀释法，测定了β-内酰胺酶抑制剂舒巴坦与氨苄西林联用对产酶耐药菌株的体外抗菌活性，并从诱导产超广谱β-内酰胺酶菌株O78?许昌F和O78?荥阳R中，制备提取出超广谱β-内酰胺酶，观察了超广谱β-内酰胺酶对三代头孢的水解作用和舒巴坦对三代头孢的抑酶保护作用。体外抑菌实验结果表明：一代头孢头孢氨苄对标准菌株的最小抑菌浓度（minimal inhibitory concentration，MIC）值在0.125－8μg/ml之间，对临床分离菌株的MIC值在0.156－8μg/ml之间，对临床菌株和标准菌株的抗菌活性基本一致；三代头孢曲松和头孢噻呋对各种兽医临床分离菌株和标准菌株的MIC值基本一致，分别在0.00625－0.06μg/ml和0.00625－0.8μg/ml之间。阿莫西林、氨苄西林则不同：对标准菌株的抗菌活性在0.03125－4μg/ml之间，对临床分离致病菌的抗菌活性在0.0625－≥500μg/ml之间；对标准菌株的抗菌活性明显强于其对临床分离菌株的活性，尤以大肠杆菌分离株最明显：阿莫西林和氨苄西林对鸡大肠杆菌标准菌株O78的MIC值分别是2μg/ml和4μg/ml，但对临床大肠杆菌分离株的MIC值却都≥500μg/ml。头孢氨苄在兽医临床应用不多，阿莫西林和<WP=7>氨苄西林已广泛应用很多年，可得出结论：细菌耐药性的产生，与抗生素的连续使用有关；同时，该体外抑菌试验结果，也很好的证明了三代头孢对临床分离致病菌的强抑菌、杀菌作用。对氨苄西林严重耐药菌大肠杆菌分离株的β-内酰胺酶鉴定检测结果：Nitrocefin纸片法检测结果，100％为阳性产酶菌株；碘测量法结果阳性株28株（阳性率97.3％）；酸测定法结果阳性株27株（阳性率97％），且不同检测方法结果差异不显著（P>0.05）。说明猪鸡常见致病菌中的大肠杆菌多产β-内酰胺酶，产生β-内酰胺酶是这些细菌对阿莫西林、氨苄西林耐药的主要原因。但对这些分离菌株用纸片协同法检测超广谱β-内酰胺酶（Extendβ-lactamase，ESBLs）的结果，却全部呈现阴性，没有一株细菌是产ESBLs菌株。产酶菌株对三代头孢头孢曲松、头孢噻呋体外抑菌试验敏感，MIC值在0.025－0.05μg/ml和0.25－0.5μg/ml之间，证明三代头孢对普通β-内酰胺酶有很好的抵抗作用。广谱β-内酰胺酶抑制剂舒巴坦对鸡大肠标准菌株O78的最小抑菌浓度值为250μg/ml，表明舒巴坦对鸡大肠杆菌标准菌株无抑菌作用；但当舒巴坦与氨苄西林联合应用时，氨苄西林对那些对其已产生严重耐药性的猪鸡常见致病菌的最小抑菌浓度MIC值，由1000μg/ml降到25μg/ml，敏感性提高了40倍，且大部分耐药菌株重新转化为敏感菌株。说明是舒巴坦具有抑酶保护作用，扩大了氨苄西林的抗菌谱，增强了氨苄西林的抗菌活性。本论文结果结合成本和价格，兽医临床提倡舒巴坦与氨苄西林以1:4的配比使用。 <WP=8>对临床分离株O78?许昌、O78?荥阳分别用三代头孢头孢曲松和头孢噻呋作底物，以亚抑菌浓度（头孢曲松0.0125μg/ml、头孢噻呋0.125μg/ml）诱导后，经检测确证产生了ESBLs。该结果说明，随着三代头孢的广泛应用，在兽医临床迟早会出现产超广谱β-内酰胺酶的菌株，这应该引起临床用药注意。这些产超广谱β-内酰胺酶的菌株，体外抑菌试验结果，仍表现出对三代头孢头孢曲松和头孢噻呋敏感，说明细菌对三代头孢的体外抑菌试验可能不反应其真实的敏感、耐药情况。舒巴坦对头孢曲松、头孢噻呋的抑酶保护作用试验中，ESBLs酶粗体液与头孢曲松和头孢噻呋作用1h后，对标准鸡大肠杆菌O78的MIC值有所下降，说明ESBLs能水解三代头孢，但MIC值仍≤2.0μg/ml，说明ESBLs菌株对三代头孢的体外抑菌试验，仍表现敏感。由头孢噻呋作底物诱导的菌株O78?许昌F，当菌体质量为0.284g时，舒巴坦与头孢曲松联用可使MIC值降低2倍，但当菌体质量增到0.3g时，舒巴坦的抑酶保护作用不明显。说明舒巴坦的抑酶保护作用与细菌产生的β-内酰胺酶量有关，当产酶量过大，舒巴坦的抑酶保护作用不能很好的得以发挥，而呈“类饱和”的现象。更多还原

【Abstract】 In order to overcome drug resistance of theβ-lactamases producing bactera, in the paper, detection of β-lactamases in bactera from swine and avain and primary study for their inhibitor,sulbactam were done.At first, for the familiarβ-lactam antibiotics ,microbial sensitivity were carried out with two fold dilution methord. Further detection of the β-lactamases from the resisting-Ampicillin strains were done with different methods: Iodimetric analysis, Racinage and Nitrocefin-based test.At the same time, the minimal inhibitory concentrations(MICs) of sulbactam combining Ampillin to the β-lactamase producing strains were measured; Extendβ-lactamase(ESBLs) were prepared from the induced O78?许昌F和O78?荥阳R,which have been induced by Ceftiofur and Ceftriaxone respectively;the action of ESBLs hydrolyzing β-lactam antibiotics and sulbactam inhibiting ESBLs were observed in the last.The results of antimicrobial susceptibility in vitro showed that: the MICs of Cephalexin to the standard strains was in the range of 0.125－8μg/ml,to the clinical strains was 0.156－8μg/ml.It has the same susceptibility to the standard and clinical strains. The third Cephalosporin have potent activity against all kinds of clinical and standard strains: the MICs of Ceftriaxone to the bacteria were in the range of 0.00625－0.06μg/ml; Ceftiofur were 0.00625－0.8μg/ml.But to Ampicillin and Amoxicillin,the results were different ,most of clinical E.colis were resisted to them,the MICs were more than 500μg/ml.From the above ,we can draw the conclusion: bacterial resistance were related to the frequency of antibiotics used .Also, the third Cephalosporin can kill and inhibit clinical bacteria commendably.The results of the detection ofβ-lactamases indicated that the total clinical E.colis have Producedβ-lactamases. At the same time,combination of sulbactam with Amoxicillin,which can induce the MIC for twenty times from 500μg/ml to 12.5μg/ml.So we can say thatthe main cause of the resistance to the β-lactam antibiotics shoud be related to Producingβ-lactamases.Additionally ,the better proportion of sulbactam and Amoxicillin shoud be 1:4 in the veterinary <WP=59>clinic,if the cost were considerated.From the test,no one bacterium produced ESBLs currently,but it produced ESBLs after induced with the third Cephalosporin. We can infer that the clinical strains will produce ESBLs in the future ,with the use of the third Cephalosporin.But the results of microbial sensitivity in vitro were susceptibile to to the third Cephalosporin.In order to gain the best therapeutic effect ,we shoud detect the ESBLs first in the veterinary clinic.更多还原