嗜麦芽窄食单胞菌I类整合子相关基因的检测分析

摘 要 目的：了解嗜麦芽窄食单胞菌临床株中Ⅰ类整合酶基因及相关的sulⅠ 和qacEΔ1基因的携带情况。 方法: 琼脂稀释法测定嗜麦芽窄食单胞菌对磺胺甲噁唑的敏感性。 PCR 扩增检测intⅠ1基因、 sulⅠ 和qacEΔ1 基因。 结果: 嗜麦芽窄食单胞菌对磺胺甲噁唑的耐药率为12.73%，intⅠ1、qacEΔ1-sulⅠ基因的阳性率分别为9.09%、7.27%，多数阳性株对磺胺甲噁唑呈高MIC。结论：嗜麦芽窄食单胞菌对磺胺甲噁唑耐药性增加可能与Ⅰ类整合子的存在有关。     

ICU分离大肠埃希菌磺胺类相关耐药基因研究

目的:了解ICU分离大肠埃希菌磺胺类耐药基因流行特征及其相关转座元件的分布情况.方法:ICU分离大肠埃希菌239株,采用K-B法分析其耐药性PCR扩增sul1、sul2、sul3,对于sul阳性株进一步扩增相关转座元件基因.结果:大肠埃希菌对磺胺异噁唑和复方新诺明的耐药率分别为80.8％、60.7％. 46株磺胺敏感株中,sul1阳性2例在193株磺胺耐药株中,检出sul1阳性120例,sul2阳性132例,sul3阳性11例；有10株三种sul基因均扩增阴性在122株sul1阳性菌中,112例Ⅰ类整合酶阳性,4例ISCR1阳性.在132株sul2阳性菌株中,5例ISCR2阳性在11株sul-3阳性菌中,9例Ⅰ类整合酶阳性,7例IS26阳性结论:ICU分离大肠埃希菌磺胺类耐药基因主要以sul2和sul1为主;Ⅰ类整合酶、ISCR1、ISCR2、IS26等转座元件与磺胺类耐药基因共存,加剧了磺胺类耐药性的播散.     

儿童与成人金黄色葡萄球菌分离株Ⅰ类整合子调查分析

摘要：目的?调查金黄色葡萄球菌中I类整合子携带情况和所携带I类整合子可变区基因分布。方法?收集临床分离金黄色葡萄球菌600株，分别分离自儿童447株和成人153株，用BD Phoenix 100进行细菌鉴定和药物敏感性试验；PCR扩增Ⅰ类整合子；用限制性片段长度多态性分析和Sanger测序技术进行基因盒分析，并比较分析不同分群分离菌株基因盒携带情况及与耐药性的关系。结果?600株金黄色葡萄球菌中236株检出Ⅰ类整合酶基因，检出率为39.3%；检出14种基因盒并组成13种组合形式，可介导氨基糖苷类抗菌药物、氯霉素、甲氧苄啶及季铵盐耐药。成人与儿童Ⅰ类整合酶和整合子可变区阳性检出率差异无统计学意义，dfrA12基因盒在儿童分离株检出率高于成人分离株(P<0.05)。儿童与成人分离株在甲氧苄啶、环丙沙星和红霉素的耐药率差异有统计学意义(P<0.05)。儿童与成人分离株均表现出携带耐药基因盒的菌株对相关抗菌药物的耐药率更高。结论?儿童与成人分离金黄色葡萄球菌Ⅰ类整合子及可变区检出率较高，并参与相应药物耐药；儿童分离菌株所携带磺胺类耐药基因盒高于成人分离株。     

多重耐药鲍曼不动杆菌相关耐药基因的分析

目的研究多重耐药鲍曼不动杆菌(MDR-AB)的耐药性,并分析其相关耐药基因tetA,tetB,sul1,dfrA,mdfA。方法 2012年8月至2013年3月江苏省镇江市第一人民医院住院患者的痰液标本中分离得到的MDR-AB菌株50株。应用纸片扩散法检测对抗菌药物的敏感度。采用聚合酶链反应(PCR)检测耐药基因tetA,tetB,sul1,dfrA1,mdfA。结果 50株MDRAB对头孢哌酮/舒巴坦、氨苄西林/舒巴坦、米诺环素、复方磺胺甲噁唑的耐药率分别为38%、64%、40%、50%,对其他参与试验的8种常见抗菌药物的耐药率均大于或等于94%。50株MDR-AB中检测到tetB、sul1和mdfA基因的表达,阳性率分别为70%、95%和45%。结论该研究中MDR-AB多重耐药情况比较严重,MDR-AB对四环素、复方磺胺甲噁唑类抗菌药物耐药与tetB、sul1及广谱抗菌药物外排泵基因mdfA等的表达密切相关。     

腹腔感染患者大肠埃希菌超广谱β-内酰胺酶及整合子研究

目的了解腹腔感染患者大肠埃希菌超广谱β-内酰胺酶(ESBLs)及Ⅰ、Ⅱ类整合子的分布情况。方法共收集分离自腹腔感染患者的大肠埃希菌62株,纸片扩散法测定16种抗菌药物敏感性,采用纸片协同扩散法检测ESBLs表型,PCR检测blaCTX-M、blaSHV、blaTEM及Ⅰ、Ⅱ类整合子基因,整合子阳性菌株进一步检测整合子的可变区基因盒。结果大肠埃希菌对碳青霉烯类、哌拉西林/他唑巴坦100%敏感,对复方磺胺甲噁唑耐药率最高(62.9%)。30株大肠埃希菌产ESBLs,其中基因型blaCTX-M-14、blaCTX-M-3、blaTEM-1、blaCTX-M-14+TEM-1分别检出16株、4株、3株、7株。未发现携带SHV型ESBLs菌株。有40株(64.5%)大肠埃希菌携带Ⅰ类整合子,其中1株同时携带Ⅰ、Ⅱ类整合子,共扩增出7种耐药基因盒组合形式。最常见的基因盒组合为dfrA17-aadA5,其次为dfrA12-orfF-aadA2,Ⅱ类整合子携带dfrA1-sat1-aadA1。结论腹腔感染大肠埃希菌主要携带blaCTX-M-14和blaTEM-1,Ⅰ类整合子的存在非常普遍,主要介导对氨基糖苷类及磺胺类耐药。     

铜绿假单胞菌整合子基因盒的相关研究

目的对北京大学人民医院不同年度临床分离的铜绿假单胞菌进行整合子基因盒检测,分析其变化趋势及其与细菌耐药性的相关性。方法应用PCR对2006—2008年临床分离的420株铜绿假单胞菌进行整合子检测,对阳性PCR产物采用HinfⅠ内切酶作限制片段多态性(RFLP)分析进行整合子分类,并对整合子阳性株进行耐药基因盒的扩增与测序。结果 420株铜绿假单胞菌中116株(27.6%)检出Ⅰ类整合子,未检出Ⅱ、Ⅲ类整合子。对2006年及2008年的整合子阳性菌株可变区基因盒扩增得到7种不同的基因盒图谱,片段大小在710～2526bp,基因盒为介导氨基糖苷类抗生素耐药的aadB、aadA族和介导甲氧苄啶耐药的dfrA1和dhfrXVB。结论该院铜绿假单胞菌中整合子检出率随年度呈上升趋势,携带的基因盒与其耐药表型有相关性。     

耐复方新诺明嗜麦芽窄食单胞菌耐药基因检测及同源性分析

目的：探讨嗜麦芽窄食单胞菌临床分离株耐复方新诺明的耐药机制及同源性。方法2013年1月至2013年12月共分离获得126株嗜麦芽窄食单胞菌，药敏试验采用 K-B法。 PCR法检测sul1、sul2、ISCR2、I类整合子。并对I类整合子PCR扩增阳性产物进行测序分析，确定基因型， ERIC-PCR对其同源性进行分析。结果嗜麦芽窄食单胞菌对复方新诺明的耐药率为17.4％。22株耐药菌有16株sul1阳性，15株I类整合子阳性；未发现sul2、ISCR2阳性株。22株耐药株分为21个基因型。结论本院嗜麦芽窄食单胞菌耐复方新诺明与sul1和I类整合子高度相关，与 sul2、ISCR2无关，耐药菌株之间无遗传相关性。     

通辽地区大肠埃希菌耐药性与Ⅰ类整合子关系研究

目的了解通辽地区大肠埃希菌临床分离株中I类整合子分布情况及基因盒类型,初步探讨耐药与整合子之间的关系。方法收集2013年10月-2014年3月通辽市某医院的非重复菌株作为试验菌株,采用Kirby-Bauer纸片法进行药敏试验;采用PCR方法扩增Ⅰ类整合酶基因,在此基础上扩增Ⅰ类整合子可变区,对部分基因盒阳性菌株测序并分析。结果 99株大肠埃希菌临床分离株对所试23种抗生素中的13种耐药率高于50%,主要为对青霉素、磺胺类药物、头孢菌素等;Ⅰ类整合酶检出55株,检出率为55.56%;基因盒携带有36株,携带率为65.46%;共检出4种基因盒,共两种组合形式arr-3-dfrA27和dfrA17-AadA5。结论大肠埃希菌的耐药性与Ⅰ类整合子的存在有很大关系,dfrA17-AadA5是常见的基因盒组合形式。     

铜绿假单胞菌整合子Ⅰ和ISCR1分布及ERIC-PCR分型

目的研究临床分离铜绿假单胞菌的整合子Ⅰ和ISCR1的分布情况,并对其进行基因分型。方法分离临床234株铜绿假单胞菌,用WHONET5.4分析菌株药敏情况,PCR检测整合酶Ⅰ、整合子Ⅰ、ISCR1以及ISCR1携带的耐药基因。ERIC-PCR进行基因分型。结果铜绿假单胞菌对阿莫西林/克拉维酸、氨苄西林、氯霉素、头孢唑啉、米诺环素、氨苄西林/舒巴坦高度耐药,对环丙沙星、头孢他啶、头孢哌酮/舒巴坦、阿米卡星、亚胺培南、美洛培南较敏感,118株整合酶Ⅰ阳性,95株Ⅰ类整合子可变区阳性,3株ISCR1和ISCRI携带的耐药基因阳性。118株整合酶Ⅰ阳性铜绿假单胞菌分为89个基因型。结论Ⅰ类整合子广泛存在于铜绿假单胞菌中,ISCRI携带率较低,ERIC-PCR可用于临床分离铜绿假单胞菌的基因分型。     

Ⅰ类整合子在多重耐药铜绿假单胞菌中的分布与结构分析

目的 了解分离于镇江地区的铜绿假单胞菌对13种常用抗生素的耐药率;明确Ⅰ类整合子基因盒结构及其在耐药基因播散中的作用.方法 采用K-B纸片法检测71株铜绿假单胞菌的耐药率;煮沸法提取71株铜绿假单胞菌基因组DNA;PCR扩增Ⅰ类整合子基因,并通过测序分析其所携带耐药基因盒.结果 71株铜绿假单胞菌对临床常用的13种抗生素的耐药率在18.3%～77.5%不等;Ⅰ类整合子检出率为38%,包括aadB、aac(6')-Ⅱ、PSE-Ⅰ、dfrA17和aadA5 5种基因盒,其中最常见者为dfrA17和aadA5.Ⅰ类整合子阳性菌株对哌拉西林、哌拉西林/他唑巴坦、头孢曲松、头孢吡肟、头孢他啶、庆大霉素、阿米卡星、妥布霉素、左氧氟沙星、环丙沙星等10种抗生素的耐药率明显高于整合子阴性菌株.结论 不同铜绿假单胞菌临床株对13种常用抗生素的耐药率各不相同,整合子阳性菌株耐药率明显高于整合子阴性菌株,提示Ⅰ类整合子是铜绿假单胞菌多重耐药的重要因素.     

Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands

OBJECTIVES: To determine the occurrence and transmission of class 1, 2 and 3 integrons in multidrug-resistant or sulfamethoxazole-resistant Salmonella from human and animal sources and in Campylobacter spp. and Escherichia coli from broilers isolated in the Netherlands in 2004. METHODS: PCR, restriction fragment length polymorphism (RFLP) and DNA sequencing were used to detect integrase genes and gene cassettes within 234 E. coli isolates, 40 Campylobacter isolates and 228 Salmonella isolates. RESULTS: Class 1 integrons were found in 76% of the E. coli and in 43% of the Salmonella isolates. Class 2 integrons were found in 11% of the E. coli and 1% of the Salmonella isolates. No class 1 or 2 integrons were detected in the Campylobacter isolates, and no class 3 integrons were detected in any of the bacterial species examined. The 22 different integrons detected harboured 20 different gene cassettes. The cassette arrays dfrA1-aadA1 and dfrA1-sat2-aadA1 were most frequently associated with class 1 and 2 integrons, respectively. For the first time linF was found to be associated with a class 2 integron as part of the linF-sat2-aadA1 cassette. The gene cassettes found within the integrons explain only a part of the resistance profile of the isolates. Conjugation experiments demonstrated transfer of class 1 and 2 integrons. CONCLUSIONS: Our data demonstrate the importance of integrons for the occurrence and transmission of multidrug resistance. Identical predominant class 1 and 2 integrons in E. coli and Salmonella serovars indicate horizontal transfer between these species.     

Prevalence of dfr genes associated with integrons and dissemination of dfrA17 among urinary isolates of Escherichia coli in Korea

OBJECTIVES: The association of trimethoprim-resistant dfr genes with integrons was investigated in urinary Escherichia coli isolates in Korea from the last two decades. METHODS: Of 623 E. coli isolates from urine specimens, 421 trimethoprim-resistant isolates were studied for dfr genes associated with integrons. Integrase genes were amplified and the PCR products restricted using HinfI to classify integron types. Gene cassette regions for the class 1 and class 2 integrons were amplified and sequenced. PFGE was performed to determine the epidemiological relationship of E. coli isolates. RESULTS: The carriage of class 1 integrons was found to be significantly higher in trimethoprim-resistant isolates (69%) than in trimethoprim-susceptible isolates (19%). Among the trimethoprim-resistant isolates, the frequency of dfr genes associated with class 1 integrons increased sharply from 10% of the isolates during 1980-1985 to 53% during 1996-1997 and to 46% during 2001-2002. Five different dfr cassettes--dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17--were identified among the urinary E. coli isolates from the last two decades; dfrA12 was the most prevalent during 1980-1985 and dfrA17 during 1996-1997 and 2001-2002. The majority of dfr genes associated with class 1 integrons were conjugally transferable to recipient E. coli strains. The E. coli isolates that carried dfrA17 associated with class 1 integrons were found to be phylogenetically unrelated, indicating that dfrA17 was widely distributed in the different clones of E. coli. CONCLUSION: Class 1 integrons were found to be an important genetic element of resistance to trimethoprim among urinary E. coli in Korea, and the prevalence of dfrA17 was mainly due to the horizontal transfer of class 1 integrons through conjugative plasmids.     

The prevalence of trimethoprim-resistance-conferring dihydrofolate reductase genes in urinary isolates of Escherichia coli in Korea

One-hundred and twenty-two urinary isolates of Escherichia coli were studied for trimethoprim resistance. Seventy-seven (63.1%) of the 122 isolates were found to be resistant to trimethoprim. Of the 77 trimethoprim-resistant isolates, 75 dfr genes were detected in 72 isolates as follows: the dfrA17 gene was the most prevalent, being found in 27 isolates, followed by dfrA12 in 26, dfrA1 in 15, dfrA5 in four and dfrA7 in three. Southern blot and PCR mapping analysis revealed that all of the dfrA17, dfrA12, dfrA5 and dfrA7 genes were located on class 1 integrons. The dfrA1 gene inserted as a gene cassette in class 1 integrons was found in 10 of 15 isolates, and the intI2 gene of Tn7 was detected in two out of five isolates. In conjugation experiments, the dfr genes inserted in class 1 integrons were transferred to a recipient E. coli in 32 (42.7%) of the 75 dfr genes. In conclusion, the dfrA17 and dfrA12 genes were the most prevalent genes responsible for trimethoprim resistance in urinary tract isolates of E. coli from Korea and the dfr genes inserted in integrons are more widespread than those that are not related to gene cassettes.     

Prevalence and characterization of class 1 and class 2 integrons in Escherichia coli isolated from meat and meat products of Norwegian origin

OBJECTIVES: The aim of the study was to investigate the prevalence of integrons and to characterize inserted gene cassettes in Escherichia coli isolated from meat and meat products of Norwegian origin. METHODS: The strains investigated (n = 241 resistant out of 944 investigated) were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET) during the years 2000-2003. PCR and DNA sequencing were used for detection of the integrase genes and gene cassettes within the integrons. RESULTS: Integrons were detected in 43 (18%) of the 241 resistant isolates. Class 1 integrons were detected in 29 (12%) strains and class 2 integrons were detected in 14 (6%) strains. Ten different gene cassettes were detected: dfrA1, dfr2a, dfrA12, aadA1, aadA2, catB2, oxa-30, sat, sat1 and orfF. The dfrA1 + aadA1 combination was the most prevalent cassette combination, detected in 12 of 29 class 1 integrons. Twelve (of 14) class 2 integrons contained a cassette area consistent with that on Tn7, the remaining two contained the cassettes sat + sat1 + aadA1. Nearly one-third of the class 1 integrons (9 of 29) lacked the sul1 gene. Ten gene cassettes (one dfr2a, two catB2 and seven aadA1) were expressed at levels below breakpoint values normally used to classify strains as resistant. CONCLUSIONS: Integrons of class 1 or 2 were present in approximately 18% of the resistant E. coli strains investigated. Certain cassette combinations in class 1 integrons seem to be more widespread than others, like the dfrA1 + aadA1. Low-level expression of antimicrobial resistance, caused by the expression of certain gene cassettes in some integrons represents an obstacle in classifying strains as susceptible or resistant.     

多重耐药大肠埃希菌中I类整合子的研究

目的分析多重耐药大肠埃希菌中Ⅰ类整合子的流行情况和分子特性。方法对81株多重耐药大肠埃希菌用PCR法扩增细菌总DNA上的Ⅰ类整合子，对大小相同的Ⅰ类整合子进行酶切分析，对纯化后的Ⅰ类整合子作DNA测序，将DNA序列在GenBank中搜索，确定Ⅰ类整合子可变区基因盒的种类和排列。结果在48株（59．3％）细菌的总DNA上检测到Ⅰ类整合子，Ⅰ类整合子的大小约为600～3000bp，48株细菌各含1～2个Ⅰ类整合子，大小相同的Ⅰ类整合子有相同的酶切图谱，整合子中最常见的基因盒为dfr17（甲氧苄啶耐药基因）、aadA5（链霉素、大观霉素耐药基因），最主要的基因盒排列为dfr17-aadA5。结论整合子在多重耐药大肠埃希菌中广泛流行，整合子是介导细菌多重耐药性的重要分子机制。     

The role of horizontal gene transfer in the spread of trimethoprim-sulfamethoxazole resistance among uropathogenic Escherichia coli in Europe and Canada

OBJECTIVES: To describe the distribution of trimethoprim-sulfamethoxazole resistance genes and the role of horizontal gene transfer and clonal expansion in recent increases of antibiotic resistance rates among uropathogenic Escherichia coli in Europe and Canada. METHODS: We identified antibiotic resistance alleles sul1, sul2, sul3 and dfr along with type 1 and type 2 integrons among 350 uropathogenic E. coli isolates from a cross-sectional study of acute, uncomplicated, community-acquired urinary tract infections in 16 western European countries and Canada (ECOSENS). RESULTS: Trimethoprim resistance gene distributions showed no regional dependency (P = 0.84). The most common trimethoprim resistance gene was dfrA1, which occurred in 37.9% of dfr containing isolates. Similarly, the sulfamethoxazole resistance gene distributions did not vary significantly by region (P = 0.20). sul2, the most common sulfamethoxazole resistance gene, was found in 77.9% of sulfamethoxazole-resistant isolates. The distribution of type 1 and type 2 integrons varied slightly by region (P = 0.04) with type 1 integrons being the more common (85.9%). We observed 34 combinations of the sul genes, dfr genes and integron types; the most common combinations were broadly disseminated across every region examined. CONCLUSIONS: Horizontal gene transfer plays a larger role than clonal expansion in the increase of trimethoprim-sulfamethoxazole resistance levels in Europe and Canada.     

Characterization of sulphonamide resistance genes and class 1 integron gene cassettes in Enterobacteriaceae, Central African Republic (CAR)

OBJECTIVES: The aim of this study was to characterize genes encoding sulphonamide resistance and gene cassettes associated with class 1 integrons in trimethoprim-sulphamethoxazole resistant Enterobacteriaceae recovered from Bangui, Central African Republic (CAR). METHODS: We studied 78 clinical Enterobacteriaceae isolates, including 16 extended-spectrum beta-lactamases producers, 10 Salmonella and 9 Shigella, resistant to trimethoprim-sulphamethoxazole as assessed by the disc diffusion method. PCR was used to test for sul1 and sul2 genes. Class 1 integron resistance gene cassettes were characterized by directly sequencing PCR products obtained with primers recognising 5' and 3' conserved regions. RESULTS: The sul1 gene was found in 67 isolates, the sul2 gene in 72 isolates and both genes in 62 isolates, while the int1 gene was found in 74 isolates. The most prevalent dfr genes were dfrA7 (49%), dfrA1 (17%) and dfrA2d (13%). CONCLUSION: These results illustrate the wide distribution of sulphonamide and trimethoprim resistance genes among Enterobacteriaceae in Bangui (CAR).     

Dissemination of sulfonamide resistance genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica strains and relation with integrons

In 200 sulfonamide-resistant Portuguese Salmonella isolates, 152 sul1, 74 sul2, and 14 sul3 genes were detected. Class 1 integrons were always associated with sul genes, including sul3 alone in some isolates. The sul3 gene has been identified in isolates from different sources and serotypes, which also carried a class 1 integron with aadA and dfrA gene cassettes.