杭州地区不同来源气单胞菌的毒力基因分布与耐药性研究
发布时间：2018-08-30 11:41
【摘要】：目的:气单胞菌(Aeromonas)广泛存在于自然界尤其是在水环境中,能够感染鱼类、鸟类、昆虫及哺乳动物等多种动物,可导致人类肠道内感染与肠道外感染。它是我国夏季腹泻的常见病原菌之一,且该菌作为一种水源性和食源性致病菌广泛流行并常引起疫情暴发,严重威胁公众健康。气单胞菌的分布情况和耐药特征世界多个地区均有报道,但在国内研究的相关报道较少,对于浙江地区肠道内、肠道外及鱼贝类感染的气单胞菌的耐药性和毒力基因情况的了解甚少,本研究对2011年7月-2013年6月期间分离的肠道内、肠道外、鱼贝类的气单胞菌进行19个毒力基因分布及抗菌药物耐药性进行研究,并对不同来源菌株进行分组比较,对毒力毒力基因与耐药的相关性进行探讨,以便为临床控制和治疗气单胞菌感染提供参考,为气单胞菌毒力基因与耐药性的研究奠定基础。方法:收集2012年5月-2013年9月期间浙江大学医学院附属第一医院的门诊及急诊急性腹泻病人的粪便标本;2011-2013年间本院各临床科室送检的肠道以外部位感染患者的引流液、胸腹水、胆汁、痰液等住院患者的标本;2011-2013年间海鲜农贸市场购买零售贝类(扇贝、花蛤、蛏子等)标本,并对各标本进行气单胞菌的分离,采用何种方法鉴定?鉴定及菌株保存。采用纸片扩散法(K-B法)进行药敏检测,采用PCR进行菌株的19种毒力基因的检测,并对肠道内、肠道外、鱼贝类感染感染气单胞菌的结果进行比较,对毒力毒力基因与耐药性的相关性进行探讨。结果:1.本研究共从腹泻患者分离到73株气单胞菌,从肠道外感染者分离到77株,从贝类的标本共分离到79株。生化表型分类显示肠道内气单胞菌以豚鼠气单胞菌(37株,50.6%)为主,其次为嗜水气单胞菌(28.8%)和温和气单胞菌(17.8%);而肠道外气单胞菌主要为嗜水气单胞菌(38株,49.3%)、豚鼠气单胞菌(33.8%);环境来源气单胞菌中温和气单胞菌占56.9%、杀鲑气单胞菌占36.7%;2.肠道内气单胞菌检出率最高的毒力基因为fla基因(63株,86.3%),其次为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%),检出率较低的基因为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%),没有检测到tapA基因;3.肠道外气单胞菌检出率最高的毒力基因为gcat基因(70株,90.9%),其次为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%),检出率较低的基因为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.鱼贝类气单胞菌气单胞菌检出率最高的毒力基因为gcat基因(70株,88.6%),其次为 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%)检出率较低的基因为aerA基因(4株,5.1%),ascV基因(7株,8.9%%),没有检测到tapA基因;5.act基因、ascF-G基因、ascV基因、gcat基因、ahyB基因、aopP基因在肠道内、肠道外及鱼贝类气单胞菌中的检出率没有显著性差别;而aerA基因、hlyA基因、lip基因在肠道外来源气单胞菌株中的检出率显著高于肠道内及鱼贝类感染气单胞菌;ascv2基因和aexT基因在鱼贝类感染气单胞菌株中的检出率显著高于肠道内与肠道外来源气单胞菌株中的检出率;6.肠道内来源气单胞菌对氨苄西林-舒巴坦和阿莫西林-克拉维酸的耐药率最高分别为90.41%和86.30%,而对哌拉西林-它唑巴坦的耐药率较低为9.59%。对头孢唑啉(第一代头孢菌素)耐药率高达76.71%,而对头孢呋辛(二代头孢菌素);头孢噻肟、头孢曲松、头孢他啶(三代头孢菌素)及头孢吡肟(四代头孢菌素)的耐药率均低于或等于9.59%。对环丙沙星的耐药率为15.07%,对左氧氟沙星的耐药率为8.22%。对头孢西丁(54.79%)和四环素(42.47%)耐药率较高外,对其他类抗菌药物的耐药率均较低;7.肠道外来源气单胞菌对氨苄西林-舒巴坦和阿莫西林-克拉维酸的耐药率最高分别为97.40%和96.10%,而对哌拉西林-它唑巴坦的耐药率为31.17%。对头孢唑啉(第一代头孢菌素)耐药率高达88.31%,而对头孢呋辛(二代头孢菌素)耐药率也高达41.56%,且明显高于肠道内来源气单胞菌的耐药率;肠道外来源气单胞菌对头孢噻肟(35.06%)、头孢曲松(35.06%)、头孢他啶(28.57%)(三代头孢菌素)及头孢吡肟(18.18%)(四代头孢菌素)的耐药率也均较高,对其他类抗菌药物的耐药率均较高,且明显高于肠道内来源气单胞菌的耐药率;8.环境来源气单胞菌对氨苄西林-舒巴坦和阿莫西林-克拉维酸的耐药率最高分别为67.09%和69.62%,而对哌拉西林-它唑巴坦的耐药率较低为2.53%。对头孢唑啉(第一代头孢菌素)耐药率高达60.76%,而对头孢呋辛(二代头孢菌素);头孢噻肟、头孢曲松、头孢他啶(三代头孢菌素)及头孢吡肟(四代头孢菌素)的耐药率均低于或等于7.59%。除对阿米卡星(24.05%)、头孢西丁(18.99%)和四环素(16.46%)耐药率较高外,对其他类抗菌药物的耐药率均较低;9.肠道内、肠道外与、鱼贝类感染的气单胞菌对青霉素类及第一代头孢菌素类(头孢唑啉)均具有很高的耐药率(60.76%);肠道外感染气单胞菌对二代头孢菌素(头孢呋辛,41.56%)、三代头孢菌素(头孢噻肟(35.06%)、头孢曲松(35.06%)、头孢他啶(28.57%))、四代头孢菌素(头孢吡肟(18.18%))、环丙沙星对及左氧氟沙星的耐药率则明显高于肠道内感染的菌株及鱼贝类感染菌株;环境来源气单胞菌对阿米卡星的耐药率显著高于肠道内及肠道外感染菌株;10.ahyB、lip、Exu、eprCAL、fla等基因的阳性菌株对二代头孢菌素(头孢呋辛)、三代头孢菌素(头孢噻肟、头孢曲松、头孢他啶)、左氧氟沙星、环丙沙星、复方新诺明等药物的耐药率均高于毒力阴性菌株的耐药率;而laf、aexT、ascF-G等基因的阳性菌株对二代头孢菌素(头孢呋辛)、三代头孢菌素(头孢噻肟、头孢曲松、头孢他啶)、左氧氟沙星、环丙沙星等药物的耐药率均低于毒力阴性菌株的耐药率。结论:1.本地区肠道气单胞菌主要为豚鼠气单胞菌,而肠道外气单胞菌主要为嗜水气单胞菌和豚鼠气单胞菌;环境来源气单胞菌以温和气单胞菌和杀鲑气单胞菌为主;2.gact、act、fla、ahyB基因在不同来源气单胞菌中普遍广泛存在;aerA、hlyA、lip基因在肠道外感染气单胞菌株相对较高,ascv2和aexT基因在鱼贝类感染菌株相对较高;3.对于青霉素类及一代头孢菌素,不同来源气单胞菌均普遍耐药;肠道外感染气单胞菌对二三四代头孢菌素、环丙沙星对及左氧氟沙星的耐药率相对较高;鱼贝类感染的气单胞菌对阿米卡星的耐药率相对较高;4.ahyB、lip、Exu、eprCAL、fla基因对二三代头抱菌素、左氧氟沙星、环丙沙星、复方新诺明的耐药可能有促进作用;而laf、aexT、ascF-G基因对二、三代头孢菌素、左氧氟沙星、环丙沙星的耐药可能有抑制作用。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R378
本文编号：2212963
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R378
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相关期刊论文 前2条
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