全耐铜绿假单胞菌40种耐药相关基因的研究

目的 研究全耐铜绿假单胞菌40种耐药相关基因.方法 采用法国生物梅里埃公司的API鉴定条/PSE5.0药敏条和美国BD公司的Phoenix NMIC/ID-109鉴定/药敏板鉴定和细菌药敏试验,应用PCR法检测 1株全耐铜绿假单胞菌临床分离株29种β-内酰胺酶相关基因、外膜蛋白D_2基因(oprD_2)、6种氨基糖苷类修饰酶基因(AMEs)、消毒剂/磺胺耐药基因(qacE△1-sull)、3种整合子基因(intⅠ 1、2、3)等40种耐药相关基因,分析其分布情况.结果 在该株菌,6种耐药相关基因阳性,两种β-内酰胺酶基因(blaTEM、bla()XA10)、两种氨基糖苷类修饰酶基因[(aac(6')-Ⅱ、aac(3)-Ⅱ)]、(qacE△1-sull和Ⅰ类整合子基因(int Ⅰ 1)],同时oprD_2缺失;其他27种β-内酰胺酶基因、4种氨基糖糖类修饰酶幕因[(aac(6')-Ⅰ b、aac(3)-Ⅰ、ant(3")-Ⅰ、ant(2")-Ⅰ)]和两种整合子基因(int Ⅰ 2、int Ⅰ 3)均为阴性.结论 该株全耐菌耐药机制为多重机制,主要与7种耐药相关基因(blaTEM、blaOXA10、oprD_2缺失、aac(6')-Ⅱ、aac(3)-Ⅱ、qacE△1-sull和Ⅰ类整合子)有关.     

儿童肠球菌多重耐药与Ⅰ类整合子的检测

目的了解儿童临床分离肠球菌的耐药特征及其多重耐药与Ⅰ类整合子的相互关系。方法采用琼脂稀释法测定常用抗菌药物对152株肠球菌的最低抑菌浓度（MIC）,用聚合酶链反应（PCR）检测肠球菌Ⅰ类整合子和Ⅰ类整合酶基因。结果屎肠球菌对氨苄西林、阿莫西林-克拉维酸、环丙沙星的耐药率分别为96．8％、95．2％和84．1％,粪肠球菌对上述3种抗菌药物的耐药率分别为23．6％、18．0％和49．4％,屎肠球菌的耐药率明显高于粪肠球菌（P〈0．001）;粪肠球菌中有2株对万古霉素的MIC为8μg/mL,粪肠球菌和屎肠球菌对替考拉宁均敏感。儿童多重耐药肠球菌发生率高达93．7％。屎肠球菌耐药模式以耐氨苄西林、红霉素、环丙沙星、利福平、四环素、高水平庆大霉素6种抗菌药物为主,占屎肠球菌的59％,粪肠球菌以耐四环素、红霉素、利福平、氯霉素、高水平庆大霉素5种抗菌药物为主,占粪肠球菌的26％。全部152株肠球菌未检测到Ⅰ类整合子,仅有5株检测到Ⅰ类整合酶基因。结论儿童肠球菌多重耐药十分严重,儿童肠球菌多重耐药与Ⅰ类整合子和Ⅰ类整合酶基因尚无明显关系。     

整合子-基因盒系统与临床细菌多重耐药的关系

细菌由不耐药转变为耐药尤其是转变为多重耐药是如今临床上面对的严峻现状.耐药机制的产生有多种原因,目前国内外与之研究较多的是整合子系统,整合子是捕获外源基因并使之转变为功能性基因的表达单位,通过转座子和接合质粒在细菌中传播.整合子（integron）包括一个特定的重组位点,里面可以插入的整合与编码某些功能的基因,被称为基因盒（genecassette）.本文将对细菌的多重耐药与整合子-基因盒的关系进行综述.     

一株多药耐药铜绿假单胞菌整合子基因结构分析

目的 检测临床分离的72株多药耐药铜绿假单胞菌(PAE)整合子分布情况,并对其中一株vim2基因阳性菌株整合子结构及定位进行分析.方法 通过药敏试验检测铜绿假单胞菌的MIC,以PCR扩增多药耐药PAE的vim2耐药基因和Ⅰ类整合子,电泳胶回收DNA片段并测序,将测序结果以Pubmed在线软件BLAST进行序列比对分析.结果 72株非重复多药耐药铜绿假单胞菌的intI基因阳性率高达99.0％,在一株多药耐药铜绿假单胞菌(命名为PAE vim2)中检测到双整合酶,两整合酶及其相连的不同基因盒均位于细菌染色体上.其中一个结构为int Ⅰ -sul1,位于Tn21样转座子中,另一个为Tn402样的Ⅰ类整合子,后者携带vim2等基因盒.结论 多药耐药PAE整合子的携带率高,且在一株多药耐药铜绿假单胞菌中发现双整合酶耐药结构,可能是引起耐药基因水平转移的遗传物质基础.     

Molecular characterization of Ambler class A to D β-lactamases,ISAba1, and integrons reveals multidrug-resistant Acinetobacter spp. isolates in northeastern China

The prevalence of various Ambler class A to D β-lactamases, ISAba1, and class 1 and 2 integrons as well as the clonal relatedness in 105 Acinetobacter spp. isolates found in northeastern China was investigated. All 105 Acinetobacter spp. isolates were determined to be multidrug resistant (MDR), and the resistance rates to carbapenem agents were approximately 50%. PER, IMP, AmpC, and OXA-23 were found to be dominant β-lactamases belonging to different classes, respectively. This is the first report of the coexistence of bla_PER, bla_IMP, bla_AmpC, and bla_OXA-23-like genes in Acinetobacter spp. isolates from northeastern China. ISAba1 was found upstream of the bla_OXA-23-like gene in 87.8% (36/41) strains and upstream of the bla_OXA-51-like gene in 26.5% (13/49) strains. ISAba3–like element was found upstream of the bla_OXA-58-like gene in one bla_OXA-58-like-positive strain. The presence of IntI1 was detected in 63.8% (67/105) of the isolates and the most prevalent gene cassettes were aacA4, aadA1, and catB8. The highly prevalent isolates belong to international clonal lineage (ICL)-II. These results indicate that the wide horizontal and clonal spread of MDR Acinetobacter spp. isolates harbouring multiple β-lactamase genes has become a serious problem in northeastern China.     

An outbreak of fluoroquinolone-resistant Pseudomonas aeruginosa ST357 harboring the exoU gene

Antimicrobial-resistant isolates of Pseudomonas aeruginosa collected from 2005 to 2014 in a university hospital in Kyoto, Japan, were retrospectively analyzed by multilocus sequence typing (MLST), exoenzyme genotype determination, integron characterization, and clinical associations. During the study, 1573 P. aeruginosa isolates were detected, and 41 of these were resistant to more than two classes of antimicrobial agents. Twenty-five (61.0%) isolates were collected from urine. All isolates were resistant to ciprofloxacin, 8 (19.5%) isolates showed resistance to imipenem/cilastatin, and 8 (19.5%) isolates showed resistance to meropenem. None of the isolates fulfilled the clinical criteria for multidrug-resistant P. aeruginosa. All isolates were negative in the metallo-β lactamase test. Thirty-six (87.8%) isolates were of the exoS^?exoU⁺ genotype and 5 (12.2%) isolates were of the exoS⁺exoU^? genotype. Among 36 exoS^?exoU⁺ isolates, 33 (80.5%) were ST357, and 3 (7.3%) were ST235. Five isolates of exoS⁺exoU^? were ST186, ST244, ST314, ST508, and ST512. Thirty-three isolates were positive for class 1 integrons and four different class 1 integrons were detected: aminoglycoside (2′) adenyltransferase and chloramphenicol transporter (AadB+CmlA6), OXA-4 β-lactamase and aminoglycoside 3′-adenyltransferase (OXA4+AadA2), AadB alone, and aminoglycoside acetyltransferase alone (AacA31). Among the 41 patients from which the isolates originated, the most common underlying disease was cancer in 16 patients (39%), and 9 patients (22.0%) died during the hospitalization period. There was no statistical correlation between MLST, exoenzyme genotype, and patient mortality. The results indicated outbreaks of fluoroquinolone-resistant P. aeruginosa in immunocompromised patients mainly due to the propagation of potentially virulent ST357 isolates possessing the exoU⁺ genotype.     

携带新型耐药基因盒组合形式Ⅰ类整合子的铜绿假单胞菌耐药特性分析

目的分析1株临床分离携有多重耐药基因盒整合子的铜绿假单胞菌的耐药特性。方法应用PCR-限制性片段长度多态性（PCR—RFLP）对整合子进行检测及分型，用长片段PCR（Long—PCR）扩增整合子的可变区并进行DNA测序，分析整合子可变区含有的耐药基因。同时应用PCR扩增金属酶基因和oprD2基因。结果该株铜绿假单胞菌PA27携带Ⅰ类整合子，其可变区大小为3.0kb，含有编码对氨基糖苷类、氯霉素和β内酰胺类抗生素耐药的基因，经与GenBank数据库进行同源性分析（BLAST），确定为一种新型基因盒组合形式：aac（6’）-Ⅱ-cm1A8-OXA-10，GenBank登录号为EU708817。oprD2基因检测阳性，未检出IMP-1、VIM-2和IMP-2型金属酶基因。结论该地区首次报道携带新型基因盒组合形式的Ⅰ类整合子的铜绿假单胞菌，并携有多重耐药基因，应引起临床高度重视。     

鲍曼不动杆菌整合子与耐药性的相关性研究

目的调查鲍曼不动杆菌中Ⅰ类、Ⅱ类和Ⅲ类整合子的存在情况,分析整合子与鲍曼不动杆菌耐药性的关系。方法测定93株鲍曼不动杆菌对抗菌药物的敏感性;应用简并引物PCR方法,同时扩增整合子5’保守区的I类、Ⅱ类和Ⅲ类整合酶基因,对阳性PCR产物用限制性内切酶Hinf I作限制片段长度多态性（RFLP）分析进行整合子分类。结果 22株鲍曼不动杆菌检测出Ⅰ类整合子,未检出Ⅱ类和Ⅲ类整合子。Ⅰ类整合子阳性的菌株对抗菌药物的耐药率普遍比整合子阴性的菌株高。结论鲍曼不动杆菌临床分离株的耐药性强,细菌的多重耐药性与Ⅰ类整合子的存在相关。     

不同地区铜绿假单胞菌第一类整合子和整合子相关基因盒的分布

目的对石家庄、昆明两地分离的铜绿假单胞菌临床菌株进行第一类整合子的分布及整合子相关基因盒携带率的比较分析，探讨第一类整合子在两地菌株分布的差别。方法采用PCR方法筛选检测64株石家庄、昆明两地临床分离的铜绿假单胞菌第一类整合子和及其所携带的整合子相关基因盒。结果64株铜绿假单胞菌临床菌中共有43株为第一类整合子阳性菌（阳性率67．2％）；其中石家庄菌株和昆明菌株阳性率分别为71．4％和64．3％，两地铜绿假单胞菌第一类整合子阳性率无显著性差异。第一类整合子阳性铜绿假单胞菌中，石家庄菌株和昆明菌株携带一类整合子相关基因盒的阳性率分别为80．0％和38．％，两地第一类整合子阳性菌中整合子相关基因盒的携带率具有显著性差异（P〈0．01）；两地菌株携带的基因盒大小不同。结论第一类整合子在铜绿假单胞菌临床菌株中广泛分布，环境的选择压力影响细菌整合子相关基因盒的分布。