动物源大肠杆菌、沙门氏菌和金黄色葡萄球菌耐药表型与氯霉素类抗菌药物耐药基因型的研究

发布时间：2018-09-06 09:22

【摘要】：目的:大肠杆菌广泛存在于自然界中,多数情况下以正常肠道菌群存在于人和动物中。现今大肠杆菌作为畜禽对抗生素耐药性的重要指示菌之一。沙门氏菌病是公共卫生学上具有重要意义的人畜共患病之一,目前发现血清型多达两千多种。动物中,鸡为沙门氏菌主要感染的源头,严重时可导致其死亡。金黄色葡萄球菌为自然界中分布最为广泛的化脓性细菌之一,可导致人或动物局部脓肿、败血症、脓毒血症等。近十几年来,为了预防和治疗细菌性疾病,促进动物的发育及生长,大量的抗菌药物应用于集约化养殖场或个体养殖户中。抗菌药物长期、广泛、不合理的使用,以及抗菌药物的选择压力等原因促使大肠杆菌、沙门氏菌和金黄色葡萄球菌耐药菌株不断增加,耐药谱不断扩大,甚至有些菌株对尚未用于临床治疗的新型抗菌药物也表现出了耐药性。氯霉素类抗菌药物是一类广谱类抗生素,由于氯霉素毒性较强,2002年我国把氯霉素列为兽用禁药,氟苯尼考作为新一代氯霉素类抗菌药物被广泛地使用于动物细菌性疾病上。因此,对动物源大肠杆菌、沙门氏菌以及金黄色葡萄球菌耐药菌株、耐药表型的流行情况以及氯霉素类抗菌药物耐药基因的分布情况特征进行研究,并分析相关性,对临床兽医及公共卫生学有着积极的意义,为遏制大肠杆菌、沙门氏菌和金黄色葡萄球菌耐药性的发展,指导兽医临床用药上有重要意义,并为进一步研究氯霉素类抗菌药物的耐药机制奠定了一定的基础。方法:本试验从2014年9月到2016年3月,在重庆市北碚区、合川区、荣昌区和垫江区等地的13个养殖场(户)分别采集大肠杆菌、沙门氏菌和金黄色葡萄球菌样品,通过革兰氏染色观察形态学特征、PCR方法检测鉴定分离大肠杆菌、沙门氏菌和金黄色葡萄球菌。采用纸片扩散法,测定最终所分离鉴定的大肠杆菌、沙门氏菌和金黄色葡萄球菌对30种抗菌药物的敏感性。再运用PCR技术,检测大肠杆菌、沙门氏菌和金黄色葡萄球菌的氯霉素类抗菌药物的耐药基因:flor、clm A、fex A、fex B、cat、pex A和cfr7种基因。分析综合所有菌株的耐药表型与耐药基因型之间的关系与符合率。结果:最终在重庆市北碚区、合川区、荣昌区和垫江县等部分地区采集大肠杆菌样品1003份,分离大肠杆菌695株,分离率为69.3%,其中鸡源393株、猪源194株、牛源108株;沙门氏菌样品867份,分离沙门氏菌66株,分离率为7.6%,其中鸡源31株、猪源26株、牛源9株;金黄色葡萄球菌样品926份,共分离103株,分离率为11.1%,其中鸡源64株、猪源29株、牛源7株;共计菌株总数为864株。药敏结果显示,大肠杆菌、沙门氏菌和金黄色葡萄球菌对30种抗菌药物均呈现不同程度的耐药。其中大肠杆菌的平均耐药率在6.2%~91.4%之间,大肠杆菌对青霉素G、氨苄西林等4种抗生素的耐药率超过了70%;对氧氟沙星、阿米卡星耐药率在20%以下。沙门氏菌的平均耐药率在9.1%~100.0%之间,沙门氏菌对青霉素G、氨苄西林等5种抗生素的耐药率均在85%以上;对氧氟沙星、头孢他啶、阿米卡星的耐药率在20%以下。金黄色葡萄球菌的平均耐药率在12.6%~79.6%之间,金黄色葡萄球菌对青霉素G、氨苄西林等4种抗生素的耐药率在70%以上;对头孢他啶、头孢唑啉等8种抗生素的耐药率不超过20%。大肠杆菌、沙门氏菌和金黄色葡萄球菌均呈现出不同程度的多重耐药,大肠杆菌多重耐药最高为29耐的菌株2株,最低为0耐的菌株1株;沙门氏菌多重耐药最高为27耐的菌株1株,最低为4耐的菌株1株;金黄色葡萄球菌多重耐药最高为25耐的菌株1株,最低为2耐的菌株有3株。从不同地区采集分离的大肠杆菌对30种抗菌药物的的耐药率来看,垫江县分离菌株的耐药率普遍高于北碚区、合川区和荣昌区;沙门氏菌不同地区对不同抗菌药物呈现不同程度的耐药;金黄色葡萄球菌,北碚区分离菌株的耐药率普遍低于合川区、荣昌区和垫江县。不同动物源大肠杆菌的耐药率,其中鸡源普遍高于猪源,猪源普遍高于牛源;不同动物源沙门氏菌的耐药率分布情况与大肠杆菌相似,鸡源耐药率最高,猪源次之,牛源耐药率最低;不同动物源金黄色葡萄球菌除对个别几类抗生素的牛源、兔源的耐药率稍高以外,鸡源的耐药率普遍高于其他几类。从不同季节采集分离大肠杆菌的耐药率得出,春季耐药率高于夏季,秋季耐药率高于夏季,冬季耐药率再次稍微降低;沙门氏菌中,菌株对21种抗菌药物的耐药率高达100.0%,夏季、秋季、冬季间耐药率没有太大差异。不同季节分离的金黄色葡萄球菌四季间耐药率的变化与大肠杆菌相似。耐药基因电泳结果显示,695株大肠杆菌中检测出flor基因408株,检出率为58.7%;clm A基因403株,检出率为58.0%;cat基因257株,检出率为37.0%;cfr基因6株,检出率为0.9%。66株沙门氏菌中检测出flor基因45株,检出率为68.2%;clm A基因41株,检出率为62.1%;cat基因52株,检出率为78.8%;cfr基因2株,检出率为3.0%。金黄色葡萄球菌中检测出flor基因28株,检出率为27.2%;clm A基因33株,检出率为32.0%;cat基因45株,检出率为43.7%;cfr基因1株,检出率为1.0%;fex A基因43株,检出率41.7%。本试验未检测到fex B和pex A基因。大肠杆菌、沙门氏菌和金黄色葡萄球菌检测出耐药基因的菌株的耐药表型与耐药基因型符合率均超过50%,其中大肠杆符合的菌株数为356株,符合率为59.6%;沙门氏菌符合的菌株数为58株,符合率为93.5%;金黄色葡萄球菌的菌株数为51株,符合率为67.1%。大肠杆菌、沙门氏菌和金黄色葡萄球菌未检测出耐药基因的菌株的耐药表型与耐药基因型符合率,其中大肠杆菌符合的菌株数为68株,符合率为69.4%;沙门氏菌符合的菌株数为2株,符合率为50.0%;金黄色葡萄球菌符合的菌株数为9株,符合率为33.3%。结论:本试验菌株样品采集时间为2014年9月至2016年3月,分别从重庆市北碚区北龙公司屠宰场、重庆市长水禽业发展有限公司、重庆市合川区奶牛养殖户陈兆强和重庆市荣昌区“重庆市种猪场”等13个养殖场采集大肠杆菌、沙门氏菌和金黄色葡萄球菌菌株样品。大肠杆菌分离率为69.3%、沙门氏菌分离率为7.6%、金黄色葡萄球菌分离率为11.1%。大肠杆菌、沙门氏菌和金黄色葡萄球菌对30种抗菌药物呈现不同程度的耐药,大部分菌株存在多重耐药的现象。且均对青霉素G、万古霉素等4种抗生素的耐药程度最为严重,耐药率较高,均超过70%;对阿米卡星耐药率较低,低于20%。大肠杆菌、沙门氏菌和金黄色葡萄球菌对flor、clm A和cat基因的检出率相对较高,其中clm A为携带菌株数最多的耐药基因。未在本试验中检测出fex B和pex A基因。大肠杆菌、沙门氏菌和金黄色葡萄球菌检测出耐药基因的菌株与其耐药表型的符合率、以及未检测出耐药基因的菌株与其耐药表型的符合率均未达到100.0%,
[Abstract]:OBJECTIVE: Escherichia coli (E.coli) is widely distributed in nature, and most of them are found in humans and animals. Today, E.coli is one of the important indicators of antibiotic resistance in livestock and poultry. S. aureus is one of the most widely distributed pyogenic bacteria in nature, which can cause local abscess, sepsis, sepsis and so on. In recent decades, in order to prevent and treat bacterial diseases, promote animal development and sepsis. A large number of antibiotics are used in intensive farms or individual farmers. The long-term, widespread, unreasonable use of antibiotics, as well as the pressure of antibiotic selection and other reasons for the increasing resistance of Escherichia coli, Salmonella and Staphylococcus aureus strains, drug resistance spectrum continues to expand, and even some strains have not yet been used. Chloramphenicol antibiotics are a kind of broad-spectrum antibiotics. Because of the strong toxicity of chloramphenicol, chloramphenicol was listed as a veterinary prohibited drug in 2002. Flufenicol, as a new generation of chloramphenicol antibiotics, is widely used in animal bacterial diseases. To study the epidemic situation of drug-resistant strains of Escherichia coli, Salmonella and Staphylococcus aureus, as well as the distribution characteristics of chloramphenicol-resistant genes, and to analyze the correlation, is of positive significance to clinical veterinary and public health, and to control E.coli, Salmonella and Staphylococcus aureus. The development of bacterial resistance is of great significance in guiding clinical veterinary drug use and laying a foundation for further study on the mechanism of chloramphenicol resistance. Methods: From September 2014 to March 2016, 13 farms (households) in Beibei District, Hechuan District, Rongchang District and Dijiang District of Chongqing City were selected to collect colic poles respectively. Samples of Escherichia coli, Salmonella and Staphylococcus aureus were identified by Gram staining and PCR. The susceptibility of 30 antibiotics to Escherichia coli, Salmonella and Staphylococcus aureus was determined by disk diffusion method. Resistance to chloramphenicol antibiotics in Escherichia coli, Salmonella and Staphylococcus aureus: flor, CLM A, Fex A, Fex B, cat, PEX A and CFR were detected by PCR. The relationship and coincidence rate between resistance phenotype and resistance genotype were analyzed and synthesized. A total of 1003 isolates of Escherichia coli and 695 isolates of Escherichia coli were isolated from Rongchang and Dianjiang counties. The isolation rate was 69.3%, of which 393 were chicken *, 194 from pig origin, 108 from cattle, 867 from Salmonella, 66 isolates from Salmonella, and 7.6% *, 31 of chicken origin, 26 from pig origin, 26 from bovine, and Staphylococcus aureus samples. A total of 103 isolates were isolated, with a separation rate of 11.1% * of which 64 strains were from chicken, 29 from pigs and 7 from cows. The total number of strains was 864. Drug sensitivity test showed that Escherichia coli, Salmonella and Staphylococcus aureus showed different degrees of resistance to 30 kinds of antibiotics. The average drug resistance rate of E. coli was between 6.2%~91.4% and E. coli to Penicillium. The resistance rates of Salmonella spp. to penicillin G, ampicillin and other antibiotics were between 9.1% and 100.0%. The resistance rates of Salmonella spp. to penicillin G, ampicillin and other five antibiotics were above 85%. The resistance rates to ofloxacin, ceftazidime and amikacin were below 20%. The average resistance rate of Staphylococcus aureus to penicillin G, ampicillin and other four antibiotics was more than 70%. The resistance rate of Staphylococcus aureus to ceftazidime, cefazolin and other eight antibiotics was less than 20%. Escherichia coli, Salmonella and Staphylococcus aureus showed different degrees of resistance. There were 2 strains with the highest 29-resistant E.coli and 1 strain with the lowest 0-resistant E.coli, 1 strain with the highest 27-resistant Salmonella and 1 strain with the lowest 4-resistant Salmonella, 1 strain with the highest 25-resistant Staphylococcus aureus and 3 strains with the lowest 2-resistant Staphylococcus aureus. The resistance rate of Enterobacter spp. to 30 kinds of antibiotics was generally higher in Dinjiang County than in Beibei, Hechuan and Rongchang districts; Salmonella showed different degrees of resistance to different antibiotics in different districts; Staphylococcus aureus and Beibei isolates were generally lower than in Hechuan, Rongchang and Dinjiang districts. The resistance rates of Escherichia coli from different animal sources were generally higher than that of pig * *, and the source of swine was generally higher than that of cattle. The distribution rate of Salmonella isolates from different animal sources was similar to E.coli * the highest resistance rate was found in chickens, followed by pigs, and lowest in cattle. The drug resistance rate of E. coli isolated from different seasons was higher in spring than in summer, higher in autumn than in summer, and slightly lower in winter. In autumn, there was no significant difference in the resistance rate in winter. The resistance rate of Staphylococcus aureus isolated in different seasons was similar to that of Escherichia coli. Among 66 strains of Salmonella spp., 45 strains of flor gene were detected, the detection rate was 68.2%; 41 strains of CLM A gene were detected, the detection rate was 62.1%; 52 strains of cat gene were detected, the detection rate was 78.8%; 2 strains of CFR gene were detected, the detection rate was 3.0%. 28 strains of flor gene were detected in Staphylococcus aureus, the detection rate was 27.2%; 33 strains of CLM A gene was detected, the detection rate was 32.0%; 45 strains of t gene, 43.7%; 1 strain of CFR gene, 1.0%; 43 strains of Fex A gene, 41.7%. The coincidence rate was 59.6% for 356 strains, 93.5% for Salmonella, and 67.1% for Staphylococcus aureus. Escherichia coli, Salmonella and Staphylococcus aureus did not detect resistant genes, and the coincidence rate between resistant phenotypes and resistant genotypes was 59.6%. The coincidence rate was 69.4%. Salmonella coincidence rate was 50.0%. Staphylococcus aureus coincidence rate was 33.3%. The isolation rate of Escherichia coli, Salmonella and Staphylococcus aureus was 69.3%, Salmonella was 7.6%, Staphylococcus aureus was 11.1%. Bacteria, Salmonella and Staphylococcus aureus were resistant to 30 kinds of antibiotics in varying degrees, and most of them were multidrug resistant. The resistance to penicillin G, vancomycin and other four kinds of antibiotics was the most serious, and the resistance rate was higher than 70%; the resistance rate to amikacin was lower than 20%. The detection rates of flor, CLM A and cat genes in S. aureus and S. aureus were relatively high, among which CLM A was the most resistant gene. The coincidence rates of resistant strains and their resistant phenotypes were not 100%.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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