规模养鸭场细菌群落结构分析及病原菌耐药性研究
发布时间：2019-06-08 18:53
【摘要】：近年来,贵州省大力发展三穗鸭养殖,给当地养殖户带来了经济效益。但养殖过程中大量鸭群排泄物给鸭场及周围环境造成了污染,同时,环境细菌也会影响鸭群健康。临床上长期使用抗生素,导致细菌大面积耐药,给养殖业带来了难题。为了解鸭场环境细菌种类及鸭群发病原因,本试验对贵州省三穗县部分鸭场环境土壤、水样中细菌种类进行研究,采用传统方法及PCR方法对患鸭内脏器官进行细菌分离鉴定,同时开展分离菌耐药性调查、耐药基因检测及耐药质粒消除等试验研究,以期为养鸭过程中环境病原微生物的消毒控制及细菌性疫病的防治提供理论依据。1.规模养鸭场环境细菌群落结构分析:采集贵州省部分鸭场环境土壤样本及水样样本,采用试剂盒提取样本中细菌总DNA,PCR扩增细菌16S rDNA V3区,对扩增产物进行DGGE电泳,最后采用Quantity One软件分析DGGE图谱,并对目的条带切胶回收并测序分析。结果显示:(1)鸭场环境土壤中共有5个细菌种类,变形菌门(Proteobacteria)的α,β类群、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、厚壁菌门(Firmicutes)及非培养细菌,其中厚壁菌门条带最多,占44.19%;其次为变形菌门,占20.93%;拟杆菌门和放线菌门均占6.98%;酸杆菌门最少,占2.33%;非培养细菌,占18.60%。(2)鸭场环境水样中共有3个细菌种类,变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、酸杆菌门(Acidobacteria)和非培养细菌,其中变形菌门条带最多,占56.67%;其次为厚壁菌门,占10.00%;酸杆菌门最少,占6.67%;其余为非培养细菌,占26.67%。2.规模养鸭场细菌分离鉴定及致病性试验:采集贵州省部分鸭场患鸭内脏器官,采用鲜血营养琼脂培养基进行细菌分离培养,挑取可疑菌落纯化培养后进行生化鉴定或PCR鉴定;随机选取主要分离菌进行致病性试验。结果显示:患鸭内脏器官中共分离到7个属137株细菌,包括埃希氏菌56株,占总分离菌的40.88%(56/137);葡萄球菌47株,占总分离菌的34.31%(47/137);里氏杆菌14株,占总分离菌的10.22%(14/137);变形杆菌9株,占总分离菌的6.57%(9/137);耶尔森菌和枸橼酸杆菌均4株,均占总分离菌的2.92%(4/137);假单胞菌3株,占总分离菌的2.19%(3/137)。致病性结果显示:埃希氏菌分离菌、葡萄球菌分离菌均可致死BALB/c鼠,致死率分别为23.3%(7/30)和16.7%(5/30);里氏杆菌分离菌则不可致死BALB/c鼠,致死率为0(0/9)。3.规模养鸭场病原菌耐药性调查及控制研究:选取15株埃希氏菌分离菌、15株葡萄球菌分离菌、3株里氏杆菌分离菌,采用药敏纸片扩散法进行耐药性分析,并对埃希氏菌E coil 1进行耐药基因检测和耐药质粒消除研究。结果表明:在20种抗生素中,埃希氏菌分离菌对10种抗生素耐药率高达100%(15/15),葡萄球菌分离菌对4种抗生素敏感率高达100%(15/15),而里氏杆菌分离菌对80%的抗生素耐药率均高于50%。埃希氏菌分离菌中可检出TEM型和CTX-M型耐药基因,检出率分别为46.7%(7/15)和100%(15/15),SHV型耐药基因未检出。通过高温-SDS交替处理,埃希氏菌E coil 1的耐药质粒出现丢失,且最佳条件为0.5%的SDS与43℃高温交替处理至第7次。综上所述,贵州省部分鸭场环境土壤中主要有变形菌门、拟杆菌门、放线菌门、酸杆菌门、厚壁菌门及非培养细菌,环境水样中主要有变形菌门、厚壁菌门、酸杆菌门及非培养细菌;患鸭内脏器官中分离到埃希氏菌、葡萄球菌、里氏杆菌等7个属137株细菌;主要分离菌对BALB/c鼠致病性程度不同;主要分离菌对试验抗生素存在广泛耐药;埃希氏菌E coil 1耐药质粒经高温-SDS交替处理后被消除。
[Abstract]:In recent years, the development of three-ear duck breeding in Guizhou province has brought economic benefits to the local farmers. However, in the process of culture, a large amount of duck group excrements cause pollution to the duck farm and the surrounding environment, and meanwhile, the environmental bacteria can also influence the health of the duck group. The long-term use of antibiotics in the clinic leads to the large-area drug resistance of the bacteria, and has brought the problem to the breeding industry. In ord to understand that bacterial species and the cause of the duck group in the duck farm, this experiment study the bacterial species in the environmental soil and water sample of part of the duck farm in the three-spike county of Guizhou province, and the traditional method and the PCR method are used to identify the bacteria in the duck's internal organs, and the drug resistance investigation of the isolate is carried out. In order to provide a theoretical basis for the control of the environmental pathogenic microorganisms and the prevention and treatment of the bacterial blight in the process of duck breeding, the drug-resistant gene detection and the elimination of drug-resistant plasmids were studied. The structure analysis of the environmental bacterial community in the scale-raising duck farm is as follows: the sample of the soil sample and the water sample in the part of the duck farm in Guizhou is collected, the total DNA of the bacteria in the sample is extracted by the kit, the 16S rDNA V3 region of the bacteria is amplified by PCR, the amplification product is subjected to DGGE electrophoresis, and the DGGE map is analyzed by using the Quantity One software, And the purpose strip is cut and recovered and sequenced and analyzed. The results showed that: (1) There were 5 bacteria species, Proteobacteria, bacteria, Bacteroides, Acinetobacter, Acidobacter, Firmick and non-cultured bacteria in the duck farm environment. 44.19% and 20.93% in the second, and 6.98% for both the Bacteroides and the actinomycetes; the minimum of the bacteria was 2.33%, and the non-cultured bacteria accounted for 18.60%. (2) There were 3 kinds of bacteria, Proteobacteria, Firmicin, Actinidia and non-cultured bacteria in the water sample of the duck farm, among which, the most of the strains were in the strain, accounting for 56.67%, the second was the thick-walled fungus, accounting for 10.00%, and the number of the acid bacteria was at least 6.67%. The rest were non-cultured bacteria, accounting for 26.67%. The isolation and identification of the bacteria in the duck farm of the scale and the pathogenicity test are as follows: the internal organs of the duck in the duck farm in Guizhou province are collected, and the bacteria are separated and cultured by using the blood nutrient agar medium, and the suspicious colony is selected and cultured for biochemical identification or PCR identification; The main isolates were randomly selected for pathogenicity test. The results showed that there were 137 strains of the genus Escherichia, including 56 strains of Escherichia coli, 40.88% (56/137) of total isolates,47 strains of Staphylococci, 34.31% (47/137) of total isolated bacteria, and 14 strains of urella, which accounted for 10.22% (14/137) of total isolates, and 9 strains of Proteus proteus, accounting for 6.57% of total isolates (9/137); The total isolates were 2.92% (4/137) and 2.19% (3/137). The results showed that the isolates of E. coli and Staphylococci could kill BALB/ c mice, and the mortality rate was 23.3% (7/30) and 16.7% (5/30) respectively. The investigation and control of the drug resistance of the pathogenic bacteria in the scale-raising ducks:15 strains of E. coli,15 strains of Staphylococci and 3 strains of urella multocida were selected, and the drug-resistant analysis was carried out by the method of drug-sensitive paper diffusion. And the drug resistance gene detection and the drug resistance plasmid elimination study are carried out on the Escherichia coli E coil 1. The results showed that, in 20 kinds of antibiotics, the drug-resistance rate of E. coli isolates was as high as 100% (15/15), and that of S. aureus isolates was as high as 100% (15/15). TEM and CTX-M resistant genes can be detected in the isolates of Escherichia coli, the detection rate is 46.7% (7/15) and 100% (15/15) respectively, and the SHV-resistant gene is not detected. The drug-resistant plasmids of Escherichia coli E coil 1 were lost by alternating treatment with high temperature-SDS, and the optimum conditions were 0.5% SDS and 43 鈩，
本文编号：2495497
[Abstract]:In recent years, the development of three-ear duck breeding in Guizhou province has brought economic benefits to the local farmers. However, in the process of culture, a large amount of duck group excrements cause pollution to the duck farm and the surrounding environment, and meanwhile, the environmental bacteria can also influence the health of the duck group. The long-term use of antibiotics in the clinic leads to the large-area drug resistance of the bacteria, and has brought the problem to the breeding industry. In ord to understand that bacterial species and the cause of the duck group in the duck farm, this experiment study the bacterial species in the environmental soil and water sample of part of the duck farm in the three-spike county of Guizhou province, and the traditional method and the PCR method are used to identify the bacteria in the duck's internal organs, and the drug resistance investigation of the isolate is carried out. In order to provide a theoretical basis for the control of the environmental pathogenic microorganisms and the prevention and treatment of the bacterial blight in the process of duck breeding, the drug-resistant gene detection and the elimination of drug-resistant plasmids were studied. The structure analysis of the environmental bacterial community in the scale-raising duck farm is as follows: the sample of the soil sample and the water sample in the part of the duck farm in Guizhou is collected, the total DNA of the bacteria in the sample is extracted by the kit, the 16S rDNA V3 region of the bacteria is amplified by PCR, the amplification product is subjected to DGGE electrophoresis, and the DGGE map is analyzed by using the Quantity One software, And the purpose strip is cut and recovered and sequenced and analyzed. The results showed that: (1) There were 5 bacteria species, Proteobacteria, bacteria, Bacteroides, Acinetobacter, Acidobacter, Firmick and non-cultured bacteria in the duck farm environment. 44.19% and 20.93% in the second, and 6.98% for both the Bacteroides and the actinomycetes; the minimum of the bacteria was 2.33%, and the non-cultured bacteria accounted for 18.60%. (2) There were 3 kinds of bacteria, Proteobacteria, Firmicin, Actinidia and non-cultured bacteria in the water sample of the duck farm, among which, the most of the strains were in the strain, accounting for 56.67%, the second was the thick-walled fungus, accounting for 10.00%, and the number of the acid bacteria was at least 6.67%. The rest were non-cultured bacteria, accounting for 26.67%. The isolation and identification of the bacteria in the duck farm of the scale and the pathogenicity test are as follows: the internal organs of the duck in the duck farm in Guizhou province are collected, and the bacteria are separated and cultured by using the blood nutrient agar medium, and the suspicious colony is selected and cultured for biochemical identification or PCR identification; The main isolates were randomly selected for pathogenicity test. The results showed that there were 137 strains of the genus Escherichia, including 56 strains of Escherichia coli, 40.88% (56/137) of total isolates,47 strains of Staphylococci, 34.31% (47/137) of total isolated bacteria, and 14 strains of urella, which accounted for 10.22% (14/137) of total isolates, and 9 strains of Proteus proteus, accounting for 6.57% of total isolates (9/137); The total isolates were 2.92% (4/137) and 2.19% (3/137). The results showed that the isolates of E. coli and Staphylococci could kill BALB/ c mice, and the mortality rate was 23.3% (7/30) and 16.7% (5/30) respectively. The investigation and control of the drug resistance of the pathogenic bacteria in the scale-raising ducks:15 strains of E. coli,15 strains of Staphylococci and 3 strains of urella multocida were selected, and the drug-resistant analysis was carried out by the method of drug-sensitive paper diffusion. And the drug resistance gene detection and the drug resistance plasmid elimination study are carried out on the Escherichia coli E coil 1. The results showed that, in 20 kinds of antibiotics, the drug-resistance rate of E. coli isolates was as high as 100% (15/15), and that of S. aureus isolates was as high as 100% (15/15). TEM and CTX-M resistant genes can be detected in the isolates of Escherichia coli, the detection rate is 46.7% (7/15) and 100% (15/15) respectively, and the SHV-resistant gene is not detected. The drug-resistant plasmids of Escherichia coli E coil 1 were lost by alternating treatment with high temperature-SDS, and the optimum conditions were 0.5% SDS and 43 鈩，
本文编号：2495497
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