耐药嗜水气单胞菌毒力与六类抗菌药物耐药元件基因研究

目的 了解嗜水气单胞菌毒力基因与耐药基因的分布情况,为嗜水气单胞菌感染的治疗提供参考依据。方法 分离自2014年1月至2017年10月本院内科住院病人的10株嗜水气单胞菌;采用PCR法,对10株嗜水气单胞菌检测3种毒力基因与54种耐药基因,并对检测结果作样本聚类分析。结果 10株耐药嗜水气单胞菌3种重要的毒力基因检测结果,hlyA基因检出率为100.00%,aerA基因检出率为20.00%, rtxA基因无检出。10株耐药嗜水气单胞菌6种β-内酰胺酶基因, blaAQU与blaMOX基因表达AmpC型β-内酰胺酶,10株中有5株同时检出blaAQU与blaMOX基因,1株仅检出blaMOX基因, 6株AmpC酶三维试验均呈阳性。对10株耐药嗜水气单胞菌和1株敏感嗜水气单胞菌3种毒力基因与6类抗菌药物54种耐药元件基因检测结果作UPGMA法样本聚类分析, 10株耐药嗜水气单胞菌和1株敏感嗜水气单胞菌可分为A与B二个群。结论 10株耐药嗜水气单胞菌共检出β-内酰胺类、氨基糖苷类、喹诺酮类、磺胺类、氯霉素类等5类抗菌药物17种获得性耐药基因。7种可移动遗传元件标记基因,有5株检出1~3种可移动遗传元件标记基因。10株耐药嗜水气单胞菌3种毒力基因与54种耐药元件基因同步检测显示,每株至少检出1种毒力基因和2种耐药基因,嗜水气单胞菌对抗菌药物的耐药率上升必须严密监测。     

嗜水气单胞菌ATCC7966 luxS基因缺失株构建及特性研究

目的研究群体感应信号分子AI-2合成酶编码基因luxS对嗜水气单胞菌ATCC 7966生理特性及毒力因子的影响。方法利用同源重组原理,构建含有中间为卡那霉素抗性基因,两侧为luxS基因上、下游同源序列片段的基因敲除质粒,将构建好的质粒转化大肠埃希氏菌SM 10(λpir)感受态细胞,利用接合法将质粒转入嗜水气单胞菌ATCC 7966,通过抗性筛选、PCR和DNA测序确认嗜水气单胞菌luxS基因缺失突变株。比较野生株ATCC 7966和luxS基因缺失突变株生长速度、AI-2合成、胞外蛋白酶合成及生物膜形成的差异。结果嗜水气单胞菌luxS基因缺失突变株ΔluxS构建成功。与野生株相比,突变株生长周期延长,基本丧失合成AI-2和胞外蛋白酶的能力;突变株生物膜形成能力降低,生长24h时生物膜形成能力为野生株的14.5%,36h时生物膜形成能力为野生株的60.0%,突变株生物膜形成速度明显比野生株缓慢,结构更为疏松。结论 luxS基因参与调控嗜水气单胞菌的生长、AI-2合成和毒力因子表达,本研究为进一步研究luxS基因在嗜水气单胞菌致病性中发挥的作用奠定基础。     

2009～2013年189株嗜水气单胞菌的临床分布与耐药性

目的了解本地区临床感染嗜水气单胞菌的分布特点及耐药情况。方法对该院2009~2013年感染性患者标本中分离的嗜水气单胞菌的分布和耐药性进行回顾性分析。结果 5年间共分离到嗜水气单胞菌189株,其中肠道内感染菌株132株,肠道外感染的57株。肠道外感染的菌株多来源于肝胆疾病(20例)、血液系统肿瘤(10例)等患者的血液(26.3%)、胆汁(21.1%)等标本。嗜水气单胞菌的感染多发于夏秋季,50岁以上的中老年人。肠道内与肠道外感染的嗜水气单胞菌的耐药率存在差别,但对氨苄西林、氨苄西林/舒巴坦、头孢唑啉、阿莫西林-克拉维酸、头孢西丁均有较高的耐药率(51.1%),肠道外感染嗜水气单胞菌仅对左氧氟沙星、亚胺培南、阿米卡星、美洛培南较敏感(耐药率10%)。结论该地区嗜水气单胞菌的感染高发于夏秋季、中老年人,以肠道内感染常见,但其对抗菌药物的敏感性较好;肠道外感染多见于肝胆疾病、肿瘤等患者,耐药率较高且在不断上升。临床医师应根据感染部位区别对待,参考药敏选药并继续加强监测。     

嗜水气单胞菌双重荧光定量PCR检测方法的建立与初步应用

目的建立快速准确检测致病性嗜水气单胞菌的双重荧光定量PCR方法。方法针对嗜水气单胞菌的16S rDNA和气溶素基因aerA的序列设计特异性引物及TaqMan探针,优化双重荧光定量PCR反应条件,并结合常规PCR方法及分离培养鉴定对临床样品进行检测和对比验证。结果荧光定量PCR反应体系对质粒标准品的敏感性为10拷贝/反应,是常规PCR检测方法的100倍;该方法检测12种其他种属细菌时未出现假阳性;对实际样品的检测结果其敏感性同样高于普通PCR,定量检测目的基因的拷贝数与样品中目的菌的分离率成正比。结论本方法敏感性高,特异性好,可用于致病性嗜水气单胞菌感染的诊断、疾病防控及流行病学研究。     

嗜麦芽窄食单胞菌磺胺类耐药与整合子的相关性研究

目的探讨临床分离嗜麦芽窄食单胞菌磺胺类药物耐药与Ⅰ、Ⅱ、Ⅲ类整合子存在的关系。方法收集临床分离的51株嗜麦芽窄食单胞菌,K-B法测定12种抗菌药物的耐药情况。PCR扩增磺胺耐药基因（sulⅠ基因）和Ⅰ、Ⅱ、Ⅲ类整合子。结果 51株嗜麦芽窄食单胞菌10株表现对复方磺胺甲噁唑耐药（19.6%）,7株菌（13.7%）Ⅰ类整合子阳性,没有检测到Ⅱ、Ⅲ类整合子,12株菌（23.5%）sulⅠ阳性。结论嗜麦芽窄食单胞菌对磺胺类药物耐药可能与Ⅰ类整合子存在有关。     

气单胞属菌粪便分离株分子鉴定与耐药元件检测

目的 调查一组14株气单胞属菌的菌种分子鉴定情况,以及β-内酰胺酶类、氨基糖苷类耐药的遗传学背景。方法 14株气单胞属菌均分离自2012年1月至12月宁波市第一医院肠道门诊腹泻患者的粪便标本,再作通用引物16SrDNA测序比对判定菌种,然后用聚合酶链反应(PCR)的方法分析23种β-内酰胺酶基因、6种氨基糖苷类修饰酶基因和6种16SrRNA甲基化酶基因以及6种可移动遗传元件分子标记。结果 本组14株菌经16SrDNA测序比对,10株为嗜水气单胞菌, 水簇箱气单胞菌、温和气单胞菌、肠棕气单胞菌、斑点气单胞菌各1株。14株气单胞菌共检出5种β-内酰胺酶基因、4种氨基糖苷类修饰酶基因和3种可移动遗传元件遗传标记基因。其中4号株(嗜水气单胞菌)AQU基因是新的基因亚型,命名为AQU-2, 11号株(水簇箱气单胞菌)AQU基因也是新的基因亚型,命名为AQU-3。结论 气单胞菌属的菌种鉴定应该以分子鉴定法为准。本组14株气单胞属菌耐药严重,已呈多重耐药。     

气单胞菌中检测到新的耐药基因aac(6’)-Ib-Cr和qnrS

目的明确气单胞菌临床分离株是否存在抗生素相关耐药基因aac(6’)-Ib-Cr和qnrS,以便更好地控制耐药菌的传播。方法选择3株临床分离自腹泻患者的耐药气单胞菌,行PCR法和基因测序检测aac(6’)-Ib-Cr和qnrS等耐药基因。结果 3株中包括嗜水气单胞菌、温和气单胞菌和豚鼠气单胞菌各1株,均耐氟喹诺酮类药物,其中2株对除亚胺培南外的所有检测抗生素耐药。3株均携带aac(6’)-Ib-Cr耐药基因,其中1株同时携带qnrS2耐药基因。结论本研究属我国首次报道气单胞菌中存在aac(6’)-Ib-Cr和qnrS2耐药基因,提示气单胞菌耐药严重,耐药机制复杂,应引起临床重视。     

耐药气单胞菌中检出多种耐药基因

目的研究临床感染气单胞菌的耐药性及耐药机制,为临床治疗提供科学依据。方法选择分离自肝病患者及腹泻患者的耐药气单胞菌19株,PCR及琼脂糖凝胶电泳法检测了β-内酰胺酶TEM,OXA;质粒AmpC酶MOX／CMY,FOX,LAT／CMY及氨基糖苷类修饰酶aac(3)-Ⅰ,aac(3)-Ⅱ,aac(16’)-Ⅰ,Ant(3”)-Ⅰ等耐药基因。结果本组19株气单胞菌中,包括嗜水气单胞菌10株,温和气单胞菌6株,豚鼠气单胞菌3株。气单胞菌耐药严重,耐氨苄西林、头孢唑林及头孢美唑的100％,耐头孢曲松63．15％,但无耐亚胺培南者。检测19株气单胞菌TEM阳性11株,OXA阳性2株,质粒AmpC酶MOX／CMY阳性7株,FOX及LAT／CMY均阴性;氨基糖苷类修饰酶aac(3)-Ⅰ阳性2株,aac(3)-Ⅱ阳性2株,aac(16’)-Ⅰ阳性4株,Ant(3”)-Ⅰ阳性6例,其中1株温和气单胞菌同时检测出上述4种耐药基因。结论我国首次检测到气单胞菌的耐药基因,其中TEM型β-内酰胺酶最高,质粒AmpC酶以MOX／CMY为主,氨基糖苷修饰酶4种基因型均存在,提示气单胞菌的耐药严重,耐药机制复杂,有多种耐药基因存在,须引起重视。     

福建地区健康人携带德尔卑沙门菌的耐药特征及遗传多样性分析

目的调查分析福建地区健康人携带德尔卑沙门菌的耐药特征及遗传多样性。方法分离自福建地区健康人的德尔卑沙门菌73株,采用K-B法进行药敏试验;采用PBRT法检测18个主要质粒家族的复制子;采用常规PCR方法扩增毒力基因;采用PFGE技术进行分子分型;应用BioNumerics软件进行聚类分析;应用SPSS 17.0进行统计学分析。结果福建地区德尔卑沙门菌健康人分离株对四环素表现出一定的耐药性(耐药率28.77%),其他药物敏感率均90%;2000年后分离的菌株总耐药率高于2000年前的分离株(χ~2=6.767,P0.01),且5株多重耐药菌株均为2000年后分离;分离株中检出IncI1型(13.70%)、IncHI2型(10.96%)和IncP型(4.11%)3种质粒复制子类型,未检出毒力基因spvB,其余毒力基因均有较高携带率;毒力基因谱型26个(VP1-VP26),其中相对优势谱型为VP1:HilA-sifA-mgtC-siiE-sopB-iroN-lpfA-stn;73个分离株共分为56个PFGE带型(PT001-PT056)、6个PFGE簇(cluster A-cluster F),其中cluster E为相对优势簇。结论福建地区健康人携带的德尔卑沙门菌耐药性逐年增强且具有致病潜能。     

奶牛粪便分离的伊氏李斯特菌伊氏亚种基因组序列测定及其分子特征分析

目的 了解某奶牛场奶牛粪便中李斯特菌的携带情况及其分离株的遗传特征。方法 用ISO 11290方法分离196份奶牛粪便样本中的李斯特菌,对分离的伊氏李斯特菌进行全基因测序,用MEGA6.0构建系统发育树,网站在线比对分析伊氏李斯特菌株的毒力基因、耐药基因及前噬菌体等。结果 196份奶牛场养殖的奶牛粪便样本中有9份样本为李斯特菌阳性,其中3株为伊氏李斯特菌伊氏亚种,6株为英诺克李斯特菌,分离率分别为1.53%和3.06%。3株伊氏李斯特菌伊氏亚种分离株具有包括LIPI-1和LIPI-2在内的绝大部分致病性李斯特菌毒力相关基因,携带一个不完整的前噬菌体及22个耐药相关基因。结论 养殖场奶牛粪便中携带伊氏李斯特菌伊氏亚种。对伊氏李斯特菌伊氏亚种分离株的全基因组、毒力基因、耐药基因、前噬菌体基因等遗传特征分析,为进一步分析其致病性提供了参考。     

Resistance to antimicrobial agents, hemolytic activity and plasmids in Aeromonas species

A total of 174 Aeromonas isolates consisting of 100 strains from patients with diarrhea being mainly overseas travellers nd healthy subjects, and 74 strains from environmental sources including foods, fish, fresh water, sea water and river soil collected in the area of Tokyo Metropolis and Kanagawa Prefecture was examined for the antimicrobial resistance, presence of plasmids and hemolytic activity. Almost all the isolates (99.4%) were resistant to aminobenzyl penicillin. The isolation frequency of chloramphenicol- or tetracycline-resistant strain was low. Most environmental isolates of A. hydrophila were resistant to multiple antimicrobial agents. Thirty-seven percent of environmental isolates and 39% of human fecal ones carried plasmids. In environmental isolates, seven A. hydrophila and three A. sobria strains carried 63- to 150-kilobase pair (kb) conjugative R plasmids. Two A. hydrophila strains from both the healthy subject and domestic case with diarrhea carried 58- to 90-kb conjugative R plasmids, respectively. None of the isolates from the feces of overseas traveller's diarrhea carried the plasmid. Irrespective of the sources. A. hydrophila showed the highest hemolytic activity among three Aeromonas species. Eighty percent or more of A. hydrophila isolates were of hemolysin positive. The hemolytic titer of A. hydrophila strains from human feces was higher than that of the strains from environmental sources.     

Enteropathogenicity of Aeromonas species isolated from infants: a cohort study

The significance of Aeromonas spp. as potential enteric pathogens was evaluated in a cohort of 187 infants aged 3-18 months during a 16-week summer period. Aeromonas spp. were isolated from 14 of the 196 (7.1%) diarrhoeal episodes detected and from eight (5.2%) of 153 samples from paired asymptomatic infants. Carriage of bacterial enteropathogens excluding Aeromonas spp. was detected in a high proportion (23%) of the asymptomatic children. Almost all of the seven isolates of Aeromonas sobria were enterotoxigenic, invasive and beta-haemolytic. In contrast, none of the seven Aeromonas caviae strains had these virulence-associated characteristics. The only isolate of Aeromonas hydrophila produced cytotoxic enterotoxin and was invasive. Plasmid analysis of selected strains did not correlate with these two properties or with antibiotic resistance. Nevertheless, the latter was found in an important proportion of the isolates. The diarrhoeal episodes, in which Aeromonas spp. were detected, lasted significantly longer, i.e. 17.2 days when the strains were invasive and/or toxigenic as compared with 4.3 days (P less than 0.001) in patients harbouring strains lacking both traits. These results reinforce the need to characterise virulence determinants before assigning any pathogenic role to Aeromonas spp. isolated from faecal specimens. Our findings also suggest the need for adequate antibiotic treatment in patients with confirmed Aeromonas spp. having enterotoxigenic and/or invasive properties.     

Genetic variations in Aeromonas hydrophila isolates from clinical and environmental sources in Thailand

Aeromonas hydrophila, a widely distributed human pathogen causing a variety of diseases, can be isolated from clinical and environmental sources. Analysis in Thailand of 110 isolates of Aeromonas hydrophila by randomly amplified polymorphic DNA-PCR (RAPD-PCR) revealed one specific RAPD pattern group (G) that was associated only with strains from environmental sources. Cytotoxic activity, adhesion to epithelial cells and exoenzyme secretions of A. hydrophila were also investigated. A comparison of isolates with pattern group G with a set of isolates derived from human blood showed low induction of cytotoxicity from those with RAPD pattern group G suggesting low virulence of these strains.     

Biochemical and genetic characterization of autoagglutinating phenotypes of Aeromonas species associated with invasive and noninvasive disease

The genetic characteristics and biochemical and structural properties of a number of autoagglutinating (AA) strains of Aeromonas associated with invasive and noninvasive disease in humans and infections in animals and from environmental sources were investigated. Of 27 strains analyzed by multilocus enzyme typing and DNA hybridization studies, 25 (93%) were confirmed to belong to either hybridization group 1 (phenospecies and genospecies Aeromonas hydrophila) or 8 (phenospecies Aeromonas sobria; genospecies Aeromonas veronii). Further analysis of 19 of these strains indicated that four major groups could be identified on the basis of serologic and surface characteristics, protein and lipopolysaccharide composition, and virulence properties; these groupings held true regardless of the site of isolation or disease process involved. The major AA+ group identified was serogroup O:11, whose strains possessed an S layer, were resistant to the bactericidal activity of normal serum, and were pathogenic in mice. The results suggest a set of useful phenotypic and structural markers for identification of specific subsets of mesophilic Aeromonas involved in a wide range of infections in the animal kingdom.     

Isolation of Aeromonas species from patients with sporadic diarrhea and characterization of Aeromonas hydrophila isolates

A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998. Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A. sobria and 8 were A. caviae. The strains of A. hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups. Although all 7 strains of A. hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes. From the results of PCR assay, the tested strains of A. hydrophila were grouped into 2 hemolysin gene types of [ahh1 + ahh3 + aerA] (n = 6) and [ahh1 + aerA] (n = 1) both of which are recognized to be enteropathogenic. Five of the 7 strains of A. hydrophila belonged to serogroup O11. These results suggest that 7 strains of A. hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O-serogroup of enteropathogenic A. hydrophila in humans.     

多重聚合酶链反应快速检测嗜水气单胞菌和爱德华菌

目的根据嗜水气单胞菌株和爱德华菌株16S rDNA基因的结构特点,设计合成了二对引物XZAH3、XZAH4和XZE7b、XZE8,建立了一种同时检测鉴别嗜水气单胞菌株和爱德华菌株的多重PCR技术。试验结果表明,用这两对引物对嗜水气单胞菌和爱德华菌株进行多重PCR,嗜水气单胞菌株只扩增出361bp一条带,而爱德华菌株只扩增出576bp一条带,而对其他鱼病病原的扩增不出现任何条带,结果均为阴性;敏感性测定结果表明,该多重PCR最低能检出10pg的嗜水气单胞菌株、爱德华菌株的DNA模板。     

Aeromonas intestinal infections in the United States

To evaluate the clinical and epidemiologic aspects of aeromonas enteritis, we studied the cases of 34 persons nationwide from whom Aeromonas hydrophila had been isolated in large numbers from stool in 1984. Compared with 68 control subjects, these patients were more likely to have drunk untreated water, usually from private wells (odds ratio = 20.9; p less than 0.01). Eighteen of the isolates belonged to a single DNA-relatedness group of the eight described for Aeromonas species, but no clear correlation between illnesses in patients and any tested genotypic or phenotypic characteristic of recovered organisms was found. Gastrointestinal complaints tended to be chronic in infected adults and acute and severe in children. Nine patients had become ill after taking antimicrobial agents to which recovered Aeromonas species were resistant; 5 persons took antimicrobials to which their Aeromonas strains were susceptible and had alleviation or resolution of their gastrointestinal symptoms. These findings indicate that at least some Aeromonas strains are enteropathogenic for the normal host and that these organisms are acquired by drinking untreated water.     

对甲硝唑耐药幽门螺杆菌中rdxA基因的表达

目的 探讨对甲硝唑耐药幽门螺杆菌(Hp)株中rdxA基因表达的差异。方法 随机收集51例快速尿素酶试验阳性患者的胃窦部黏膜,体外微需氧培养。甲硝唑耐药率检测分别用E试验和临界点药敏法。选取对甲硝唑高度耐药菌株(MIC值>256 mg/L),在含16 mr/L甲硝唑及不含甲硝唑培养基中分组培养,半定量RT-PCR法检测rdxA基因的表达,用galE基因作外参照。结果 共分离出Hp临床分离菌47株。E试验法甲硝唑耐药率为34％(16/47),临界点药敏法为32％(15/47),其中15株(包括5株高度耐药菌株)2种方法均耐药。5株高度耐药菌中rdxA基因在甲硝唑存在的情况下表达量减少,而参照galE基因表达均不变。结论rdxA基因的表达减少在Hp对甲硝唑高度耐药形成中起了一定的作用。