本文選題：鴨疫里默氏桿菌 + 紅霉素抗性�。� 參考：《山東農業(yè)大學》2015年碩士論文

【摘要】：鴨疫里默氏桿菌是黃桿菌科鴨疫里默氏桿菌屬的代表種,黃桿菌科是擬桿菌門最大的一個分支,目前鴨疫里默氏桿菌已成為危害養(yǎng)鴨業(yè)的主要病原之一,不僅感染鴨、火雞,其他畜禽如雞、鵝等也容易受到感染。病鴨死亡率高、生長遲緩、飼料轉化效率低、品質下降、疾病治療等,給養(yǎng)殖業(yè)造成了巨大的經濟損失�？刂圃摬〉姆椒ㄖ饕亲⑸湟呙绾蛻每咕幬�,其中紅霉素已在臨床上成功應用于該病,但是近些年紅霉素治療失敗的案例已經被多次報道。近年來分離出來的具有紅霉素抗性的鴨疫里默氏桿菌比例逐漸增加,而鴨疫里默氏桿菌的紅霉素抗性機制還未被報道。故本試驗重點研究鴨疫里默氏桿菌對紅霉素的耐藥機制。本研究以先采用微量肉湯稀釋法分別檢測了1996年至2014年本實驗室自臨床病死鴨分離的79株鴨疫里默氏桿菌及一株鴨疫里默氏桿菌型菌株ATCC11845的最小抑菌濃度(MIC),結果表明檢測的80株鴨疫里默氏桿菌中有43株具有紅霉素抗性,占53.8%,37株為紅霉素敏感菌株,占46.2%。此外,2005—2014年自病鴨分離的56株鴨疫里默氏桿菌中有41株為紅霉素耐藥菌,占73.2%(41/56);而1996—2004年自病鴨分離的24株鴨疫里默氏桿菌中只有2株為紅霉素耐藥菌。為了確定所攜帶紅霉素基因的類型,通過PCR檢測耐藥菌株的ermF基因,結果表明,具有紅霉素抗性的43株鴨疫里默氏桿菌中有30株攜帶有ermF或ermFU基因,MIC在32~2048mg/L;具有emF+紅霉素抗性的30株菌中有27株攜帶ermFU基因,占90%(27/30);另外3株菌攜帶有ermF基因。另外,其余13株紅霉素抗性的鴨疫里默氏桿菌攜帶有ereD基因,MIC在4~16 mg/L�？寺〔煌琫rmF+陽性菌株的ermF/ermFU基因并進行序列分析,結果表明其氨基酸同源性高達98.5%-100%。對鴨疫里默氏桿菌菌株HXb2、GuiZ-1、YXb15、HGb1、NJ-4、JY-6、YZ-1、ZJb2分別通過基因組步移法擴增ermF基因及其側翼序列,結果顯示,鴨疫里默氏桿菌與CTnDOT的ermFU基因的上游序列有85.8%-88.7%的同源性,因此推測ermFU基因可能是來自CTnDOT轉座子元件;而鴨疫里默氏桿菌的ermF基因的上游(-1至-600)與轉座子Tn4351中的ermF基因相應區(qū)域相比較同源性較高,僅有幾個核苷酸的差異,因此推測ermF基因可能來自于轉座子Tn4351。此外,序列分析顯示ermF、ermFU和ereD基因存在于鴨疫里默氏桿菌基因組的多重耐藥區(qū)(MRR)。為了進一步確定ermF、ermFU和ereD基因能否引起鴨疫里默氏桿菌的紅霉素耐藥,將分別攜帶有ermF、ermFU和ereD基因盒的重組穿梭質粒轉入到紅霉素敏感菌株CH3中。結果顯示,三株重組菌CH3(pRES-HXb2-ermFU),CH3(pRES-YZ-1-erm F)和CH3(pRES-SX-ere D)均具有紅霉素抗性,其中攜帶有ermF或ermFU的兩個重組菌株CH3(pRES-HXb2-ermFU)和CH3(pRES-YZ-1-ermF)對林可霉素具有較高的抗性,而CH3(pRES-SX-ereD)對林可霉素同野生株一樣對紅霉素敏感。熒光定量PCR顯示,三株重組菌CH3(pRES-HXb2-ermFU),CH3(pRES-YZ-1-erm F)和CH3(pRES-SX-ere D)中ermFU、ermF和ereD的m RNA相對表達量分別較野生株HXb2、YZ-1和SX高3.3、80和6.7倍。以上結果表明,鴨疫里默氏桿菌多重耐藥區(qū)(DRR)中的ermF、ermFU或ereD基因可引起鴨疫里默氏桿菌的紅霉素耐藥。
[Abstract]:The Bacillus pestis are the representative species of the family of the family of the bacilliaceae, and the family of the family is the largest branch of the bacteriobacteriaceae. At present, the bacilli has become one of the main pathogens that harm the duck industry. It is not only infected with ducks, turkeys, but also other livestock and poultry such as chicken and goose. The low efficiency of feed conversion, the decline of the quality and the treatment of disease have caused great economic loss to the breeding industry. The main methods to control the disease are the injection of vaccines and the application of antibiotics. In recent years, erythromycin has been successfully applied to the disease, but in recent years the cases of erythromycin treatment have been reported many times. The ratio of erythromycin resistance to erythromycin gradually increased and the mechanism of erythromycin resistance was not reported. This study focused on the mechanism of drug resistance to erythromycin. This study was conducted from 1996 to 2014 by micro broth dilution method. The minimum inhibitory concentration (MIC) of 79 strains of M. ducks isolated from dead ducks and one strain of the strain of the strain ATCC11845 showed that 43 of the 80 strains had erythromycin resistance, 53.8%, 37 erythromycin sensitive, 56 from 2005 to 2014, and 56 strains isolated from the disease duck from 2005 to 2014. 41 strains were erythromycin resistant bacteria, accounting for 73.2% (41/56), while only 2 strains of erythromycin resistant strains isolated from 1996 to 2004 were erythromycin resistant strains. In order to determine the type of erythromycin gene carried, the ermF basis of the resistant strain was detected by PCR. The results showed that 43 strains of erythromycin resistance were found in the duck epidemic. 30 of meribacilli were carried with ermF or ermFU genes and MIC in 32~2048mg/L; 27 of the 30 strains with emF+ erythromycin resistance were carrying ermFU genes, accounting for 90% (27/30), and the other 3 strains carried ermF gene. In addition, the remaining 13 strains of erythromycin resistance were carried with ereD gene, MIC in 4~16 mg/L.. The ermF/ermFU gene of the strain and sequence analysis showed that its amino acid homology was up to 98.5%-100%. for the strain HXb2, GuiZ-1, YXb15, HGb1, NJ-4, JY-6, YZ-1, and ZJb2 respectively. The ermF gene and its flanking sequence were amplified by the genome step method respectively. The sequence has the homology of 85.8%-88.7%, so it is presumed that the ermFU gene may come from the CTnDOT transposon element, while the upstream of the ermF gene of riemulibacillus duck pestis (-1 to -600) is more homologous to the corresponding region of the ermF gene in the transposon Tn4351, with only a few nucleotide differences. Therefore, it is presumed that the ermF gene may come from the transposing. In addition, sequence analysis showed that ermF, ermFU and ereD genes existed in the multidrug-resistant region of the genome of reeminorus pestis (MRR). In order to further determine whether the ermF, ermFU and ereD genes could cause erythromycin resistance to reeminorus pestis, the recombinant shuttle plasmid containing ermF, ermFU and ereD gene boxes would be transferred to erythromycin, respectively. The results showed that three recombinant strains CH3 (pRES-HXb2-ermFU), CH3 (pRES-YZ-1-erm F) and CH3 (pRES-SX-ere D) all had erythromycin resistance, and the two recombinant strains carrying ermF or ermFU were resistant to lincomycin. The fluorescent quantitative PCR showed that the relative expression of three recombinant strains CH3 (pRES-HXb2-ermFU), CH3 (pRES-YZ-1-erm F) and CH3 (pRES-SX-ere D) was higher than that of wild plants, respectively, and 6.7 times higher than that of wild strains. F, ermFU or ereD genes can cause erythromycin resistance in R.

【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

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本文編號：1809187