整合子-基因盒系统与金黄色葡萄球菌耐药相关性的研究现状

金黄色葡萄球菌在临床革兰阳性菌感染中的检出率居首位,金黄色葡萄球菌分离株的耐药率呈逐年升高趋势,并且多重耐药菌株也在不断增加。分析和阐明金黄色葡萄球菌耐药机制对控制细菌耐药性的产生与传播至关重要。整合子-基因盒系统能够识别外源性基因盒,通过位点特异性重组捕获或切除耐药性基因盒使捕获的耐药性基因盒表达,在细菌间耐药基因的扩散和多重耐药性的产生中起到重要作用。文章主要综述了整合子-基因盒系统与金黄色葡萄球菌获得性耐药之间的关系。     

细菌基因盒-整合子系统研究进展

基因盒是小的可移动的脱氧核糖核酸DNA分子,通常整合到整合子中转录;整合子是保守、可移动的转座子样DNA元件,可以整合耐药基因在细菌之间水平传播,对于细菌间耐药基因的扩散和多重耐药性的产生起重要作用。从基因盒-整合子系统的概念提出以来,新型整合子不断被检出,研究也越来越深入,目前公认的有Ⅰ、Ⅱ、Ⅲ三类整合子,其中以Ⅰ类整合子检出最多,研究也最为细致。此文对基因盒-整合子系统的起源、结构、分类、表达、生物学检测及其与细菌耐药性之间的关系等方面研究进展进行了综述。     

整合子与细菌多重耐药性

细菌的多重耐药已成为临床治疗的难题,其耐药机制、耐药基因的传播与转移是近年来研究的热点。近来研究表明,细菌中存在一种能捕获和表达基因的遗传单位———整合子在细菌获得耐药机制中起了重要作用。整合子编码一个整合酶负责基因盒在重组位点attⅠ和attC上的插入及切除,同时整合子也提供一个启动子(Pant)负责基因盒耐药基因的表达。整合子携带着重组的基因盒插入到转座子或接合质粒中,在不同的细菌间运动而传播耐药性,同时一个整合子可以捕获多个基因盒,对细菌多重耐药的形成起重要作用。现就整合子的结构、类型、基因盒的种类与表达及其与细菌多重耐药性的有关研究进行综述。     

整合子-基因盒系统与细菌耐药性

整合子 基因盒系统在细菌中能捕获外来耐药基因,是细菌耐药性传播的机制之一。整合子携带着重组的基因盒插入到转座子或接合质粒中,在不同的细菌间运动而传播耐药性;同时一个整合子可以捕获多个基因盒,使细菌产生多重耐药性,细菌产生多重耐药性的能力取决于它们捕获新的抗生素耐药基因的能力。整合子是一种遗传因素,编码一个位点特异重组酶(IntI)负责基因盒在 attI位点的插入,同时整合子也提供一个启动子(Pant)负责基因盒耐药基因的表达。文章对整合子 基因盒的结构、种类、耐药基因盒的表达及耐药基因的获得和传播进行综述。     

猪致病性大肠杆菌耐药性与整合子-基因盒的相关性

为了解猪致病性大肠杆菌耐药性与整合子-基因盒的相关性,本研究采用腹腔注射法测定52株大肠杆菌对昆明小鼠的致病性;采用K-B法测定致病性大肠杆菌对15种抗菌药物的敏感性;对致病性大肠杆菌Ⅰ、Ⅱ和Ⅲ型整合子及其基因盒进行PCR检测及测序分析,并分析其整合子-基因盒与耐药性的相关性。结果显示,52株大肠杆菌对小鼠均具有较强致病性,96.15%分离株对15种抗菌药物表现为多重耐药性,61.54%分离株耐药性均在八重耐药以上。Ⅰ、Ⅱ和Ⅲ型整合子阳性率分别为84.62%,15.38%和3.85%。Ⅰ型整合子检测到dfrA17-aadA5(18.18%)、dfrA12-orfF-aadA2(4.55%)和dfrA1-catB3-aacA4(4.55%)基因盒,Ⅱ型整合子检测到dfrA1-sat2-aadA1(50.00%)基因盒,Ⅲ型整合子未检测到基因盒,基因盒阳性率与细菌耐药性不存在相关性。     

整合子——基因盒系统与细菌多重耐药

整合子是近年来在细菌中发现的一种可移动性基因元件。细菌通过整合子——基因盒系统能捕获外来耐药基因,在整合子中形成多种耐药基因的组合、排列,是细菌耐药性播散的机制之一,是导致耐药基因在细菌间水平播散的重要原因。就整合子的发现与分类,基因盒结构与种类,整合子对基因盒的捕获与表达,整合子与细菌耐多重耐药的关系等进行综述。     

整合子——基因量系统与细菌多重耐药

整合子是近年来在细菌中发现的一种可移动性基因元件.细菌通过整合子--基因盒系统能捕获外来耐药基因,在整合子中形成多种耐药基因的组合、排列,是细菌耐药性播散的机制之一,是导致耐药基因在细菌间水平播散的重要原因.就整合子的发现与分类,基因盒结构与种类,整合子对基因盒的捕获与表达,整合子与细菌耐多重耐药的关系等进行综述.     

整合子-基因盒系统与细菌耐药

整合子是细菌基因组中可移动的遗传物质,可以通过其整合酶将携带有耐药基因的基因盒整合到自身基因组中,并使其表达,从而使细菌获得耐药性,甚至多重耐药性.作为近年来关于细菌,尤其是革兰氏阴性菌耐药机制研究的热点,整合子一基因盒系统受到人们越来越多的关注.     

健康畜禽肠道大肠杆菌耐药性及整合子-基因盒检测

采用微量肉汤稀释法检测455株大肠杆菌对24种抗菌药物的敏感性;多重PCR检测菌株Ⅰ、Ⅱ、Ⅲ型整合酶基因;采用PCR-RFLP和PCR-测序法对整合子-基因盒进行序列分析。结果显示,455株菌除对头孢唑啉、头孢曲松、头孢噻呋和阿米卡星耐药率低于40%外,对其余药物表现出高耐药率,99.23%菌株呈多重耐药性;455株菌Ⅰ型整合子阳性率87.69%,Ⅱ型整合子1.98%,未检出Ⅲ型整合子;随机选取的204株整合子阳性菌中174株(85.29%)扩增出了基因盒可变区。基因盒主要以编码氨基糖苷腺苷基转移酶和二氢叶酸还原酶的aadA、dfrA基因为主,耐药基因排列成Ⅰ型整合子8种基因盒,Ⅱ型整合子3种基因盒。Ⅰ型整合子以dfrA1-aadA1(32.76%)基因盒为主,其次是aadA22(24.14%)和dfrA17-aadA5(24.14%)。Ⅱ型整合子以dfrA1-sat2-aadA1(66.67%)为主,Ⅱ型整合子阳性菌含有Ⅰ型整合子-基因盒;菌株含整合子-基因盒越复杂,其多重耐药性越严重。结果表明,本次分离的健康动物肠道大肠杆菌耐药非常严重,整合子-基因盒分布广泛,已成为耐药基因储库,对耐药基因扩散起重要作用。     

健康畜禽肠道大肠杆菌耐药性及整合子-基因盒检测

采用微量肉汤稀释法检测455株大肠杆菌对24种抗菌药物的敏感性;多重PCR检测菌株Ⅰ、Ⅱ、Ⅲ型整合酶基因;采用PCR-RFLP和PCR-测序法对整合子-基因盒进行序列分析。结果显示,455株菌除对头孢唑啉、头孢曲松、头抱噻呋和阿米卡星耐药率低于40％外,对其余药物表现出高耐药率,99．23％菌株呈多重耐药性;455株菌Ⅰ型整合子阳性率87．69％,Ⅱ型整合子1．98％,未检出Ⅲ型整合子;随机选取的204株整合子阳性菌中174株（85．29％）扩增出了基因盒可变区。基因盒主要以编码氨基糖苷腺苷基转移酶和二氢叶酸还原酶的aadA、dfrA基因为主,耐药基因排列成Ⅰ型整合子8种基因盒,Ⅱ型整合子3种基因盒。Ⅰ型整合子以dfrA1-aadA1（32．76％）基因盒为主,其次是aadA22（24．14％）和dfrA17-aadA5（24．14％）。Ⅱ型整合子以d厂rA1-sat2-aadA1（66．67％）为主,Ⅱ型整合子阳性菌含有I型整合子-基因盒;菌株含整合子-基因盒越复杂,其多重耐药性越严重。结果表明,本次分离的健康动物肠道大肠杆菌耐药非常严重,整合子-基因盒分布广泛,已成为耐药基因储库,对耐药基因扩散起重要作用。     

动物源沙门菌耐药性调查及Ⅰ类整合子的检测

采用肉汤微量稀释法,选用22种常用抗菌药物,对98株动物源沙门菌进行药敏试验,结果显示:菌株对磺胺类、四环素类药物及萘啶酸普遍耐药,对氨基糖苷类药物耐药率较低,对氟喹诺酮类药物高度敏感;菌株多重耐药率为67.35%(66/98).采用PCR方法检测菌株Ⅰ类整合子的流行情况,并分析其携带的耐药基因盒.结果98株沙门菌中Ⅰ类整合子的检出率为50.0%(49/98),并且均携带耐药基因盒,基因盒以dfrA17-aadA5的组合形式最为常见;Ⅰ类整合子阳性菌株的多重耐药率为95.92%(47/49),阴性菌株的多重耐药率为38.78%(19/49).上述结果表明Ⅰ类整合子普遍存在于兽医临床耐药沙门菌中,其流行与菌株多重耐药性具有一定的相关性.     

禽养殖场中季铵盐抗性菌株的分离及其I类整合子特性研究

对来自禽养殖场的季铵盐抗性菌进行分离及鉴定,并通过分析第一类整合子特性来探讨整合子对细菌耐药性的影响。生化鉴定耐药菌,采用K-B法研究菌株对10种抗生素的药敏试验,PCR法扩增一类整合酶基因和内部的基因盒,电泳检测后测序。结果显示：22株菌全部对至少3种抗生素表现出耐药性。Ⅰ类整合子检测全部阳性,检出率为100%。对其基因盒进行扩增,只有11株检测含有耐药基因盒,其序列分析显示多对甲氧苄啶、氨基糖苷类抗生素耐药。Ⅰ类整合子广泛存在于禽养殖环境菌中,其对细菌多重耐药的产生和传播起着重要作用。     

Characterization of integrons in multiple antimicrobial resistant Escherichia coli isolates from bovine endometritis

To assess the prevalence of antimicrobial resistance and three classes of integrons in Escherichia coli (E. coli) strains (n = 57) isolated from bovine endometritis in Inner Mongolia of China, antimicrobial susceptibility and the presence of three types of integrons were characterized. Most isolates were susceptible to ceftiofur, furazolidone, ciprofloxacin and enrofloxacin, while 57 isolates were all resistant to sulfamethoxydiazine and trimethoprim. High resistant incidence rates were exhibited to sulfadiazine, tetracycline, oxytetracycline, cefazolin, chloramphenicol. Forty-six of 57 E. coli strains were resistant to above 10 antibiotics (80.70%). The integrase gene and gene cassettes of integrons were amplified by PCR. DNA sequencing and analysis were used to identify the genetic content of the integron-variable regions. Neither class II nor class III integron was detected, while 36.8% (n = 21) of the isolates were positive for the presence of intI1 gene. Analysis of gene cassettes revealed that six gene cassettes were found, which encoded resistance to trimethoprim (dhfr, dhfrI, dfrA17) and aminoglycosides (aadA1, aadA2, aadA5). Among them, the gene cassette array dfrA17–aadA5 was found most prevalent (66.7%). The resistance profile of positive-integron isolates was relatively broad and they were resistant to more than eight antimicrobials (n ? 8). The correlation analysis revealed the incidence of integrons among the isolates were related to the multiple antibiotic resistance profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.     

Characterization of integrons-mediated antimicrobial resistance among Escherichia coli strains isolated from bovine mastitis

To assess the prevalence of antimicrobial resistance and class I integrons in Escherichia coli strains (n=58) isolated from bovine mastitis in Inner Mongolia, antimicrobial susceptibility and the presence of various types of integrons were characterized. Most isolates were susceptible to amikacin, colistin, ceftazidime, gentamicin and kanamycin, while those also exhibited high resistant incidence rates to ampicillin, amoxicillin, sulfadiazine and sulfamethoxydiazine. The integrase gene of integrons was amplified by PCR using degenerate primers. The integrons were confirmed by restriction fragment length polymorphism (RFLP) analysis of positive PCR products. Neither class II nor class III integron was detected, while 56.90% (n=33) of the isolates were positive for the presence of intI1 gene. Sequencing analysis of gene cassettes revealed that seven gene cassettes were found, which encoded resistance to trimethoprim (dfrA1 and dfrA17), aminoglycosides (aacA4, aadA1 and aadA5) and chloramphenicol (catB3), respectively. Of them, the gene cassette array dfrA17-aadA5 was found most prevalent (62.96%). The percentage of positive-integron among the isolates whose resistant profile was relatively broad (n> or =7) is 100.00%, while the one in narrow-profile isolates (n=2-6) is 30.56%. The correlation analysis revealed the incidence of integrons among the isolates were highly related to the resistant profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.     

Study on Molecular Characterization of Class Ⅰ Integron and Integron-associated Antimicrobial Resistance in Escherichia coli from Beef Cattle

To investigate the prevalence and molecular characterization of class Ⅰ integron in Escherichia coli isolated from beef cattle, and analyze the relationship between integron and antimicrobial resistance, susceptibilities to 11 antimicrobials were conducted on 92 isolates, the presence and characterization of class Ⅰ integrons and inserted gene cassettes were performed using PCR combined with sequencing analysis. The results showed that 29 isolates had been detected positive for class Ⅰ integron integrase gene (intⅠ1) among 92 isolates, and aadA1 and dfrA17＋aadA5 were the most prevalent gene cassette arrays detected. The resistance rates of 92 isolates to ampicillin, streptomycin, sulfisoxazole and tetracycline were all more than 45.0%. As revealed by analyzing the association between resistance phenotypes and class Ⅰ integron, isolates that contained the class Ⅰ integron were significantly highly resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole and sulfamethoxazole-trimethoprim (P<0.05), but not to quinolones and amoxicillin/clavulanic acid. The conclusion was that E.coli isolated from beef cattle were seriously resistant to antimicrobials,and integron/cassettes widely existed. The presence of integrons and the association of antimicrobial resistance determinants with transferable elements might play a crucial role in the dissemination of antibiotic resistance among E.coli. Data reported here clearly emphasized the need for a stricter application of antimicrobials restriction policies in feedlot setting.     

鸡源大肠杆菌整合子及基因盒分子流行病学研究

对河南省鸡源大肠杆菌Ⅰ类、Ⅱ类和Ⅲ类整合子及其携带的耐药基因盒进行了分子流行病学研究。结果表明：51株鸡源大肠杆菌中86．3％（44／51）检出Ⅰ类整合子,3．9％（2／51）检出Ⅱ类整合子,未检出Ⅲ类整合子。Ⅰ类整合子共检出5种类型的基因盒,分剐是sat基因盒（100％）,dfrAl＋aadAl基因盒（45．4％,20／44）,dfrAl7＋aadA5基因盒（22．5％,9／44）,dfrAl＋sat＋aadA2基因盒（6．8％,3／44）和4800bp未知基因盒（27．3％,12／44）,其中4800bp未知基因盒的下游携带有ESBLCTX-M基因,Ⅱ类整合子的基因盒携带dfrAl＋sat＋aadAl3种基因。     

Role of coresistance in the development of resistance to chloramphenicol in Escherichia coli isolated from sick cattle and pigs

OBJECTIVE: To determine the cause of persistent resistance to chloramphenicol (CP) after the ban on its use in food-producing animals in several countries. SAMPLE POPULATION: 71 CP-resistant and 104 CP-susceptible Escherichia coli strains isolated from sick cattle and pigs in Japan. PROCEDURE: Susceptibility of all bacterial strains to thiamphenicol (TP) and florfenicol (FFC) was tested by use of an agar dilution method. The CP-resistance genes and variable region within class 1 integrons in CP-resistant strains were identified by use of a PCR assay. RESULTS: The CP acetyltransferase gene (ie, cat1) was identified as the predominant CP-resistance gene in strains isolated from cattle, and the cat1and nonenzymatic CP-resistance gene (ie, cmlA) were the predominant CP-resistance genes in strains isolated from pigs. Additionally, strains with cat1 isolated from cattle often were resistant to ampicillin, dihydrostreptomycin (DSM), oxytetracycline, and trimethoprim (TMP), whereas strains with cat1 or cmlA isolated from pigs often were resistant to DSM and TMP. Class 1 integrons were significantly more prevalent in strains with CP-resistance genes, compared with prevalence in strains without CP-resistance genes. All gene cassettes within the integrons were involved in resistance to DSM, TMP, or both. CONCLUSIONS AND CLINICAL RELEVANCE: Coresistance that develops because of the use of DSM and TMP in cattle and pigs apparently contributes to the selection of CP-resistant strains of E coli. Thus, it is possible that bacterial resistance to CP in animals would persist despite a ban on the use of CP in cattle and pigs.     

Characterization of multidrug resistant Salmonella recovered from diseased animals

Three hundred and eighty Salmonella isolates recovered from animal diagnostic samples obtained from four state veterinary diagnostic laboratories (AZ, NC, MO, and TN) between 2002 and 2003 were tested for antimicrobial susceptibilities and further characterized for bla(CMY) beta-lactamase genes, class 1 integrons and genetic relatedness using PFGE. Forty-seven serovars were identified, the most common being S. Typhimurium (26%), S. Heidelberg (9%), S, Dublin (8%), S. Newport (8%), S. Derby (7%), and S. Choleraesuis (7%). Three hundred and thirteen (82%) isolates were resistant to at least one antimicrobial, and 265 (70%) to three or more antimicrobials. Resistance was most often observed to tetracycline (78%), followed by streptomycin (73%), sulfamethoxazole (68%), and ampicillin (54%), and to a lesser extent chloramphenicol (37%), kanamycin (37%), amoxicillin-clavulanic acid (20%), and ceftiofur (17%). With regards to animal of origin, swine Salmonella isolates displayed the highest rate of resistance, being resistant to at least one antimicrobial (92%), followed by those recovered from turkey (91%), cattle (77%), chicken (68%), and equine (20%). Serovars commonly showing multidrug resistance (MDR) to > or =9 antimicrobials were S. Uganda (100%), S. Agona (79%), and S. Newport (62%), compared to S. Heidelberg (11%) and S. Typhimurium (7%). Class-1 integrons were detected in 43% of all isolates, and were found to contain aadA, aadB, dhfr, cmlA and sat1 gene cassettes alone or in various combinations. All ceftiofur resistant isolates (n=66) carried the bla(CMY) beta-lactamase gene. A total of 230 PFGE patterns were generated among the 380 isolates tested using XbaI, indicating extensive genetic diversity across recovered Salmonella serovars, however, several MDR clones were repeatedly recovered from different diseased animals.