【摘要】：目的:氣單胞菌(Aeromonas)廣泛存在于自然界尤其是在水環(huán)境中,能夠感染魚類、鳥類、昆蟲及哺乳動物等多種動物,可導致人類腸道內(nèi)感染與腸道外感染。它是我國夏季腹瀉的常見病原菌之一,且該菌作為一種水源性和食源性致病菌廣泛流行并常引起疫情暴發(fā),嚴重威脅公眾健康。氣單胞菌的分布情況和耐藥特征世界多個地區(qū)均有報道,但在國內(nèi)研究的相關報道較少,對于浙江地區(qū)腸道內(nèi)、腸道外及魚貝類感染的氣單胞菌的耐藥性和毒力基因情況的了解甚少,本研究對2011年7月-2013年6月期間分離的腸道內(nèi)、腸道外、魚貝類的氣單胞菌進行19個毒力基因分布及抗菌藥物耐藥性進行研究,并對不同來源菌株進行分組比較,對毒力毒力基因與耐藥的相關性進行探討,以便為臨床控制和治療氣單胞菌感染提供參考,為氣單胞菌毒力基因與耐藥性的研究奠定基礎。方法:收集2012年5月-2013年9月期間浙江大學醫(yī)學院附屬第一醫(yī)院的門診及急診急性腹瀉病人的糞便標本;2011-2013年間本院各臨床科室送檢的腸道以外部位感染患者的引流液、胸腹水、膽汁、痰液等住院患者的標本;2011-2013年間海鮮農(nóng)貿(mào)市場購買零售貝類(扇貝、花蛤、蟶子等)標本,并對各標本進行氣單胞菌的分離,采用何種方法鑒定?鑒定及菌株保存。采用紙片擴散法(K-B法)進行藥敏檢測,采用PCR進行菌株的19種毒力基因的檢測,并對腸道內(nèi)、腸道外、魚貝類感染感染氣單胞菌的結果進行比較,對毒力毒力基因與耐藥性的相關性進行探討。結果:1.本研究共從腹瀉患者分離到73株氣單胞菌,從腸道外感染者分離到77株,從貝類的標本共分離到79株。生化表型分類顯示腸道內(nèi)氣單胞菌以豚鼠氣單胞菌(37株,50.6%)為主,其次為嗜水氣單胞菌(28.8%)和溫和氣單胞菌(17.8%);而腸道外氣單胞菌主要為嗜水氣單胞菌(38株,49.3%)、豚鼠氣單胞菌(33.8%);環(huán)境來源氣單胞菌中溫和氣單胞菌占56.9%、殺鮭氣單胞菌占36.7%;2.腸道內(nèi)氣單胞菌檢出率最高的毒力基因為fla基因(63株,86.3%),其次為gcat基因(60 株,82.2%),Exu 基因(50 株,68.5%),lip 基因(35 株,47.9%),ahyB基因(35株,47.9%),檢出率較低的基因為aerA基因(5株,6.8%),aexT基因(6 株,8.2%),ascv2 基因(7 株,9.6%),ascV 基因(8 株,11.0%),沒有檢測到tapA基因;3.腸道外氣單胞菌檢出率最高的毒力基因為gcat基因(70株,90.9%),其次為fla 基因(60 株,77.9%),lip 基因(60 株,77.9%),Exu 基因(55 株,71.4%),ahyB 基因(43 株,55.8%),eprCAL 基因(43 株,55.8%),hlyA 基因(39 株,50.6%),檢出率較低的基因為tapA基因(8株,10.4%),ascv2基因(8株,10.4%),aexT 基因(8 株,10.4%),ascV 基因(10 株,13.0%);4.魚貝類氣單胞菌氣單胞菌檢出率最高的毒力基因為gcat基因(70株,88.6%),其次為 fla 基因(53 株,67.1%),alt 基因(47 株,59.5%),lip 基因(41 株,51.9%),ahyB基因(39株,49.4%)檢出率較低的基因為aerA基因(4株,5.1%),ascV基因(7株,8.9%%),沒有檢測到tapA基因;5.act基因、ascF-G基因、ascV基因、gcat基因、ahyB基因、aopP基因在腸道內(nèi)、腸道外及魚貝類氣單胞菌中的檢出率沒有顯著性差別;而aerA基因、hlyA基因、lip基因在腸道外來源氣單胞菌株中的檢出率顯著高于腸道內(nèi)及魚貝類感染氣單胞菌;ascv2基因和aexT基因在魚貝類感染氣單胞菌株中的檢出率顯著高于腸道內(nèi)與腸道外來源氣單胞菌株中的檢出率;6.腸道內(nèi)來源氣單胞菌對氨芐西林-舒巴坦和阿莫西林-克拉維酸的耐藥率最高分別為90.41%和86.30%,而對哌拉西林-它唑巴坦的耐藥率較低為9.59%。對頭孢唑啉(第一代頭孢菌素)耐藥率高達76.71%,而對頭孢呋辛(二代頭孢菌素);頭孢噻肟、頭孢曲松、頭孢他啶(三代頭孢菌素)及頭孢吡肟(四代頭孢菌素)的耐藥率均低于或等于9.59%。對環(huán)丙沙星的耐藥率為15.07%,對左氧氟沙星的耐藥率為8.22%。對頭孢西丁(54.79%)和四環(huán)素(42.47%)耐藥率較高外,對其他類抗菌藥物的耐藥率均較低;7.腸道外來源氣單胞菌對氨芐西林-舒巴坦和阿莫西林-克拉維酸的耐藥率最高分別為97.40%和96.10%,而對哌拉西林-它唑巴坦的耐藥率為31.17%。對頭孢唑啉(第一代頭孢菌素)耐藥率高達88.31%,而對頭孢呋辛(二代頭孢菌素)耐藥率也高達41.56%,且明顯高于腸道內(nèi)來源氣單胞菌的耐藥率;腸道外來源氣單胞菌對頭孢噻肟(35.06%)、頭孢曲松(35.06%)、頭孢他啶(28.57%)(三代頭孢菌素)及頭孢吡肟(18.18%)(四代頭孢菌素)的耐藥率也均較高,對其他類抗菌藥物的耐藥率均較高,且明顯高于腸道內(nèi)來源氣單胞菌的耐藥率;8.環(huán)境來源氣單胞菌對氨芐西林-舒巴坦和阿莫西林-克拉維酸的耐藥率最高分別為67.09%和69.62%,而對哌拉西林-它唑巴坦的耐藥率較低為2.53%。對頭孢唑啉(第一代頭孢菌素)耐藥率高達60.76%,而對頭孢呋辛(二代頭孢菌素);頭孢噻肟、頭孢曲松、頭孢他啶(三代頭孢菌素)及頭孢吡肟(四代頭孢菌素)的耐藥率均低于或等于7.59%。除對阿米卡星(24.05%)、頭孢西丁(18.99%)和四環(huán)素(16.46%)耐藥率較高外,對其他類抗菌藥物的耐藥率均較低;9.腸道內(nèi)、腸道外與、魚貝類感染的氣單胞菌對青霉素類及第一代頭孢菌素類(頭孢唑啉)均具有很高的耐藥率(60.76%);腸道外感染氣單胞菌對二代頭孢菌素(頭孢呋辛,41.56%)、三代頭孢菌素(頭孢噻肟(35.06%)、頭孢曲松(35.06%)、頭孢他啶(28.57%))、四代頭孢菌素(頭孢吡肟(18.18%))、環(huán)丙沙星對及左氧氟沙星的耐藥率則明顯高于腸道內(nèi)感染的菌株及魚貝類感染菌株;環(huán)境來源氣單胞菌對阿米卡星的耐藥率顯著高于腸道內(nèi)及腸道外感染菌株;10.ahyB、lip、Exu、eprCAL、fla等基因的陽性菌株對二代頭孢菌素(頭孢呋辛)、三代頭孢菌素(頭孢噻肟、頭孢曲松、頭孢他啶)、左氧氟沙星、環(huán)丙沙星、復方新諾明等藥物的耐藥率均高于毒力陰性菌株的耐藥率;而laf、aexT、ascF-G等基因的陽性菌株對二代頭孢菌素(頭孢呋辛)、三代頭孢菌素(頭孢噻肟、頭孢曲松、頭孢他啶)、左氧氟沙星、環(huán)丙沙星等藥物的耐藥率均低于毒力陰性菌株的耐藥率。結論:1.本地區(qū)腸道氣單胞菌主要為豚鼠氣單胞菌,而腸道外氣單胞菌主要為嗜水氣單胞菌和豚鼠氣單胞菌;環(huán)境來源氣單胞菌以溫和氣單胞菌和殺鮭氣單胞菌為主;2.gact、act、fla、ahyB基因在不同來源氣單胞菌中普遍廣泛存在;aerA、hlyA、lip基因在腸道外感染氣單胞菌株相對較高,ascv2和aexT基因在魚貝類感染菌株相對較高;3.對于青霉素類及一代頭孢菌素,不同來源氣單胞菌均普遍耐藥;腸道外感染氣單胞菌對二三四代頭孢菌素、環(huán)丙沙星對及左氧氟沙星的耐藥率相對較高;魚貝類感染的氣單胞菌對阿米卡星的耐藥率相對較高;4.ahyB、lip、Exu、eprCAL、fla基因對二三代頭抱菌素、左氧氟沙星、環(huán)丙沙星、復方新諾明的耐藥可能有促進作用;而laf、aexT、ascF-G基因對二、三代頭孢菌素、左氧氟沙星、環(huán)丙沙星的耐藥可能有抑制作用。
[Abstract]:AIM: Aeromonas exists widely in nature, especially in aquatic environment. It can infect fish, birds, insects and mammals, and can cause intestinal and extraintestinal infections in humans. Aeromonas is one of the common pathogens of summer diarrhea in China, and it is widely used as a water-borne and food-borne pathogen. The distribution and drug resistance characteristics of Aeromonas have been reported in many parts of the world, but there are few reports in China. The drug resistance and virulence genes of Aeromonas isolated from intestinal tract, intestinal tract, fish and shellfish in Zhejiang Province are poorly understood. The distribution of 19 virulence genes and antimicrobial resistance of Aeromonas isolated from intestinal tract and intestinal tract of fish and shellfish from July 2011 to June 2013 were studied. Different strains from different sources were grouped and compared. The correlation between virulence genes and drug resistance was discussed in order to control and treat the infection of Aeromonas. Methods: Fecal specimens from outpatients and emergency patients with acute diarrhea in the First Affiliated Hospital of Zhejiang University Medical College from May 2012 to September 2013 were collected, and drainage fluid and thorax from patients with infections outside the intestinal tract were collected from various clinical departments during 2011-2013. Ascites, bile, sputum and other hospitalized patients'specimens; 2011-2013 seafood farmers' market to buy retail shellfish (scallops, clams, oysters, etc.) specimens, and the specimens of Aeromonas isolation, identification and preservation of strains, using disk diffusion method (K-B method) for drug sensitivity testing, using PCR strains of 19 kinds of toxins Results: 1. Seventy-three strains of Aeromonas were isolated from patients with diarrhea, 77 strains were isolated from patients with diarrhea, and 79 strains were biochemically isolated from shellfish. Phenotypic classification showed that Aeromonas in the intestinal tract were mainly Aeromonas guinea pigs (37 strains, 50.6%), followed by Aeromonas hydrophila (28.8%) and Aeromonas sobrinus (17.8%); Aeromonas hydrophila (38 strains, 49.3%) and Aeromonas guineapigs (33.8%) were the main extraintestinal aeromonas; Aeromonas sobrinus was 56.9% and salmon-killing Aeromonas environmental sources. Fla gene (63 strains, 86.3%) was the most virulent gene, followed by gcat gene (60 strains, 82.2%), Exu gene (50 strains, 68.5%), lip gene (35 strains, 47.9%), ahyB gene (35 strains, 47.9%) and aexT gene (6 strains, 8.2%) and ascv2 gene (7 strains, 9.6%). 3. The most virulent genes were gcat (70 strains, 90.9%), fla (60 strains, 77.9%), lip (60 strains, 77.9%), Exu (55 strains, 71.4%), ahyB (43 strains, 55.8%), eprCAL (43 strains, 55.8%), hlyA (39 strains, 50.6%) and hlyA (55.6%). The genes with the lowest detection rate were tapA gene (8 strains, 10.4%), ascv2 gene (8 strains, 10.4%), aexT gene (8 strains, 10.4%) and ascV gene (10 strains, 13.0%). 4. The most virulent genes were gcat gene (70 strains, 88.6%), fla gene (53 strains, 67.1%), ALT gene (47 strains, 59.5%) and lip gene (41 strains, 51.9%). AyB gene (39 strains, 49.4%) was found to be aerA gene (4 strains, 5.1%) and ascV gene (7 strains, 8.9%). tapA gene was not detected; 5. act gene, ascF-G gene, ascV gene, gcat gene, ahyB gene, aopP gene were not significantly different in intestine, intestine and outer intestine, and in Aeromonas sp. The detection rate of lip gene in the intestinal exogenous Aeromonas strains was significantly higher than that in the intestinal and fish and shellfish infected Aeromonas; the detection rate of ascv2 gene and aexT gene in the fish and shellfish infected Aeromonas strains was significantly higher than that in the intestinal and intestinal exogenous Aeromonas strains; 6. The highest resistance rates of sulbactam and amoxicillin-clavulanic acid were 90.41% and 86.30% respectively, while those of piperacillin-tazobactam were 9.59%. The resistance rates to cefazolin (first-generation cephalosporins) were 76.71%, while to cefuroxime (second-generation cephalosporins), cefotaxime, ceftriaxone, ceftazidime (third-generation cephalosporins) and ceftazidime (third-generation cephalosporins) were 76.71%. Resistance rates to cefepime (fourth generation cephalosporins) were lower than or equal to 9.59%. Resistance rates to ciprofloxacin and levofloxacin were 15.07% and 8.22%, respectively. Resistance rates to cefoxitin (54.79%) and tetracycline (42.47%) were higher, but to other antibiotics were lower; 7. The highest resistance rates of sulbactam and amoxicillin-clavulanic acid were 97.40% and 96.10% respectively, while the resistance rates of piperacillin-tazobactam were 31.17%. The resistance rates of cefazolin (first generation cephalosporins) were 88.31%, and cefuroxime (second generation cephalosporins) were 41.56%, which were significantly higher than those of enteric aeromonas. Resistance rates of enteric exogenous Aeromonas to cefotaxime (35.06%), ceftriaxone (35.06%), ceftazidime (28.57%) and cefepime (18.18%) were higher, and the resistance rates to other antibiotics were higher than those of enteric Aeromonas. The highest resistance rates to ampicillin-sulbactam and amoxicillin-clavulanic acid were 67.09% and 69.62% respectively, while the low resistance rates to piperacillin-tazobactam were 2.53%. The resistance rates to cefazolin (first generation cephalosporins) were as high as 60.76%, while to cefuroxime (second generation cephalosporins), cefotaxime and ceftriaxone (ceftriaxone). The resistance rates to ceftazidime (third generation cephalosporins) and cefepime (fourth generation cephalosporins) were lower than or equal to 7.59%. The resistance rates to other antimicrobial agents were lower except for amikacin (24.05%), cefoxitin (18.99%) and tetracycline (16.46%). Both the first and second generation cephalosporins (cefazolin) were highly resistant (60.76%), the second generation cephalosporins (cefuroxime, 41.56%), the third generation cephalosporins (cefotaxime, 35.06%), ceftazidime (28.57%), the fourth generation cephalosporins (cefepime, 18.18%), ciprofloxacin and the third generation cephalosporins (cefotaxime, 35.06%). The resistance rate of levofloxacin was significantly higher than that of intestinal infection strains and fish and shellfish infection strains; the resistance rate of environmental Aeromonas to amikacin was significantly higher than that of intestinal and extraintestinal infection strains; 10. ahyB, lip, Exu, eprCAL, fla and other gene positive strains to second generation cephalosporins (cefuroxime), third generation cephalosporins (cephalosporins) Drug resistance rates of thioxime, ceftriaxone, ceftazidime, levofloxacin, ciprofloxacin, and compound trimethoprim were higher than those of virulent negative strains, while positive strains of laf, aexT, ascF-G were more resistant to second-generation cephalosporins (cefuroxime), third-generation cephalosporins (cefotaxime, ceftriaxone, ceftazidime), levofloxacin, and ciprofloxacin. Conclusion: 1. Aeromonas in the intestinal tract is mainly Aeromonas guinea pig, while Aeromonas hydrophila and Aeromonas guinea pig are the main extraintestinal Aeromonas; Aeromonas militaris and Aeromonas salicidal are the main environmental sources; 2. gact, act, fla, ahyB Aeromonas aera, hlyA, lip genes are relatively high in the extraintestinal infection of Aeromonas strains, ascv2 and aexT genes are relatively high in fish and shellfish infection strains; 3. For penicillins and first-generation cephalosporins, Aeromonas from different sources are generally resistant; Aeromonas enterocolitica is relatively high in the 234 generations of the head. Aeromonas isolated from fish and shellfish had relatively high resistance to amikacin; 4. AhyB, lip, Exu, eprCAL, fla genes may promote the resistance of cephalosporins, levofloxacin, ciprofloxacin and compound sinomenine; and laf, aexT, ascF-G genes may promote the resistance of Aeromonas isolated from fish and shellfish to amikacin. The resistance of the three generation cephalosporins, levofloxacin and ciprofloxacin may be inhibited.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R378

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