規(guī)模養(yǎng)鴨場細(xì)菌群落結(jié)構(gòu)分析及病原菌耐藥性研究
發(fā)布時間：2019-06-08 18:53
【摘要】：近年來,貴州省大力發(fā)展三穗鴨養(yǎng)殖,給當(dāng)?shù)仞B(yǎng)殖戶帶來了經(jīng)濟(jì)效益。但養(yǎng)殖過程中大量鴨群排泄物給鴨場及周圍環(huán)境造成了污染,同時,環(huán)境細(xì)菌也會影響鴨群健康。臨床上長期使用抗生素,導(dǎo)致細(xì)菌大面積耐藥,給養(yǎng)殖業(yè)帶來了難題。為了解鴨場環(huán)境細(xì)菌種類及鴨群發(fā)病原因,本試驗對貴州省三穗縣部分鴨場環(huán)境土壤、水樣中細(xì)菌種類進(jìn)行研究,采用傳統(tǒng)方法及PCR方法對患鴨內(nèi)臟器官進(jìn)行細(xì)菌分離鑒定,同時開展分離菌耐藥性調(diào)查、耐藥基因檢測及耐藥質(zhì)粒消除等試驗研究,以期為養(yǎng)鴨過程中環(huán)境病原微生物的消毒控制及細(xì)菌性疫病的防治提供理論依據(jù)。1.規(guī)模養(yǎng)鴨場環(huán)境細(xì)菌群落結(jié)構(gòu)分析:采集貴州省部分鴨場環(huán)境土壤樣本及水樣樣本,采用試劑盒提取樣本中細(xì)菌總DNA,PCR擴(kuò)增細(xì)菌16S rDNA V3區(qū),對擴(kuò)增產(chǎn)物進(jìn)行DGGE電泳,最后采用Quantity One軟件分析DGGE圖譜,并對目的條帶切膠回收并測序分析。結(jié)果顯示:(1)鴨場環(huán)境土壤中共有5個細(xì)菌種類,變形菌門(Proteobacteria)的α,β類群、擬桿菌門(Bacteroidetes)、放線菌門(Actinobacteria)、酸桿菌門(Acidobacteria)、厚壁菌門(Firmicutes)及非培養(yǎng)細(xì)菌,其中厚壁菌門條帶最多,占44.19%;其次為變形菌門,占20.93%;擬桿菌門和放線菌門均占6.98%;酸桿菌門最少,占2.33%;非培養(yǎng)細(xì)菌,占18.60%。(2)鴨場環(huán)境水樣中共有3個細(xì)菌種類,變形菌門(Proteobacteria)、厚壁菌門(Firmicutes)、酸桿菌門(Acidobacteria)和非培養(yǎng)細(xì)菌,其中變形菌門條帶最多,占56.67%;其次為厚壁菌門,占10.00%;酸桿菌門最少,占6.67%;其余為非培養(yǎng)細(xì)菌,占26.67%。2.規(guī)模養(yǎng)鴨場細(xì)菌分離鑒定及致病性試驗:采集貴州省部分鴨場患鴨內(nèi)臟器官,采用鮮血營養(yǎng)瓊脂培養(yǎng)基進(jìn)行細(xì)菌分離培養(yǎng),挑取可疑菌落純化培養(yǎng)后進(jìn)行生化鑒定或PCR鑒定;隨機(jī)選取主要分離菌進(jìn)行致病性試驗。結(jié)果顯示:患鴨內(nèi)臟器官中共分離到7個屬137株細(xì)菌,包括埃希氏菌56株,占總分離菌的40.88%(56/137);葡萄球菌47株,占總分離菌的34.31%(47/137);里氏桿菌14株,占總分離菌的10.22%(14/137);變形桿菌9株,占總分離菌的6.57%(9/137);耶爾森菌和枸櫞酸桿菌均4株,均占總分離菌的2.92%(4/137);假單胞菌3株,占總分離菌的2.19%(3/137)。致病性結(jié)果顯示:埃希氏菌分離菌、葡萄球菌分離菌均可致死BALB/c鼠,致死率分別為23.3%(7/30)和16.7%(5/30);里氏桿菌分離菌則不可致死BALB/c鼠,致死率為0(0/9)。3.規(guī)模養(yǎng)鴨場病原菌耐藥性調(diào)查及控制研究:選取15株埃希氏菌分離菌、15株葡萄球菌分離菌、3株里氏桿菌分離菌,采用藥敏紙片擴(kuò)散法進(jìn)行耐藥性分析,并對埃希氏菌E coil 1進(jìn)行耐藥基因檢測和耐藥質(zhì)粒消除研究。結(jié)果表明:在20種抗生素中,埃希氏菌分離菌對10種抗生素耐藥率高達(dá)100%(15/15),葡萄球菌分離菌對4種抗生素敏感率高達(dá)100%(15/15),而里氏桿菌分離菌對80%的抗生素耐藥率均高于50%。埃希氏菌分離菌中可檢出TEM型和CTX-M型耐藥基因,檢出率分別為46.7%(7/15)和100%(15/15),SHV型耐藥基因未檢出。通過高溫-SDS交替處理,埃希氏菌E coil 1的耐藥質(zhì)粒出現(xiàn)丟失,且最佳條件為0.5%的SDS與43℃高溫交替處理至第7次。綜上所述,貴州省部分鴨場環(huán)境土壤中主要有變形菌門、擬桿菌門、放線菌門、酸桿菌門、厚壁菌門及非培養(yǎng)細(xì)菌,環(huán)境水樣中主要有變形菌門、厚壁菌門、酸桿菌門及非培養(yǎng)細(xì)菌;患鴨內(nèi)臟器官中分離到埃希氏菌、葡萄球菌、里氏桿菌等7個屬137株細(xì)菌;主要分離菌對BALB/c鼠致病性程度不同;主要分離菌對試驗抗生素存在廣泛耐藥;埃希氏菌E coil 1耐藥質(zhì)粒經(jīng)高溫-SDS交替處理后被消除。
[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，
本文編號：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，
本文編號：2495497
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