奇异变形菌中整合子分布及耐药性分析

目的 了解院内奇异变形菌中各类整合子的携带分布情况、阳性菌株可变区基因盒类型以及其与宿主菌耐药表型的相关性,从而为临床治疗和院内感染控制提供参考。方法 采用PCR扩增和琼脂糖凝胶电泳等方法,将本院2016年1月—2018年12月份从临床标本中分离得到的150株奇异变形菌进行第1、2和3类整合子的筛选,并对整合子阳性菌株可变区进行测序分析以及宿主菌的耐药性进行相关性分析。结果 150株奇异变形菌中携带整合子的菌株共有91株,阳性率为60.7%,其中第1类整合子阳性菌株有30株,占20.0%;第2类整合子阳性菌株22株,占14.7%;同时携带第1和2类菌株39株,占26.0%;未筛出第3类整合子;在91株整合子阳性菌株中,86株可变区出现扩增产物条带,其余5株可变区未见扩增产物;第1类整合子阳性菌株可变区携带的耐药基因盒主要为AadA2、DfrA32,第2类整合子阳性菌株可变区携带耐药基因盒主要为DfrA1;可变区携带AadA2的菌株对庆大霉素和妥布霉素的耐药率显著高于整合子阴性菌株(P<0.01),可变区携带DfrA1或DfrA32的菌株对复方磺胺甲噁唑(即甲氧苄啶/磺胺甲噁唑)的耐药率也明显高于整合子阴性菌株(P<0.01);91株整合子阳性菌株对氨苄西林/舒巴坦、复方磺胺甲噁唑、环丙沙星、庆大霉素、头孢曲松、妥布霉素和左氧氟沙星的耐药率均显著高于整合子阴性菌株(P<0.01)。结论     

辽宁地区沙门菌整合子与耐药相关性研究

摘要：目的 及时掌握辽宁地区沙门菌整合子的分布以及对常用抗菌药物的耐药情况,获取沙门菌整合子与耐药性的相关性,从而为临床用药及治疗提供指导。方法 本文对来源于食品以及病患的149株沙门菌,利用PCR扩增的方法,进行整合子类别的筛选,并将扩增的整合子基因盒进行基因测序,同时通过药敏板测定(耐药性实验)对沙门菌与15种临床常用抗菌药物的耐药相关性进行研究。结果 149株沙门菌中未发现第II类、第III类整合子,检出第I类整合子的菌株50株,I类整合子阳性率为33.6%。50株整合子阳性菌株中25株携带耐药基因盒,片段范围从1500~1800 bp。测序结果表明,其中22株整合子携带dfrA17-aadA5基因盒,2株携带dfrA12-aadA2基因盒,1株首次检出罕见耐药基因盒linG。根据整合子携带情况不同,对15种抗菌药物的耐药率进行对比分析,结果显示整合子阳性菌对9种抗菌药物的耐药率显著高于整合子阴性菌(P<0.01),分别为氨苄西林、四环素、氯霉素、头孢噻肟、头孢唑林、庆大霉素、复方磺胺甲恶唑、阿奇霉素和环丙沙星。辽宁地区沙门菌多重耐药率达50.3%。结论 I类整合子在沙门菌中分布广泛,抗性基因表型与耐药结果相一致,整合子的携带与沙门菌多重耐药率高度相关。沙门菌对亚胺培南和头孢西丁保持高敏感率,可以用于对常规抗菌药物耐药的沙门菌的治疗。     

产AmpC酶大肠埃希菌中整合子的研究

目的:观察25株产AmpC酶大肠埃希菌中整合子的分类、结构及其在介导AmpC酶基因转移中的作用。方法:采用微量稀释法测定20种抗生素对试验菌株的敏感性。利用多重聚合酶链反应(PCR)方法检测整合酶基因(intI)及其定位,对其阳性菌株可变区(Int)扩增产物进行测序分析。结果:这25株菌对多种抗生素耐药。20株Ⅰ类整合酶基因阳性(80%);所携带的耐药基因盒绝大多数为aadA5和dfr17;未发现携带AmpC基因盒的整合子。结论:Ⅰ类整合子广泛地存在于产AmpC酶的大肠杆菌中;耐药基因盒是整合子阳性菌株对氨基糖苷类、磺胺类药物及氯霉素耐药的主要原因,但对介导AmpC酶基因转移,不起主要作用。     

临床分离志贺菌耐药性分析及1类整合子的检测

目的了解2007年至2010年安徽省临床分离志贺菌耐药性和1类整合酶基因(intⅠ1)、qacE△1-sul1基因及整合子携带的耐药基因盒的分布。方法琼脂稀释法检测21种抗菌药物对志贺菌的最低抑菌浓度。PCR扩增intⅠ1和qacE△1-sul1基因,对阳性菌株可变区基因盒序列进行分析。结果 137株志贺菌对萘啶酸、氨苄西林、磺胺甲噁唑/甲氧苄啶的耐药率均80.0%以上,且多重耐药率达94.2%,但对三代头孢菌素和氟喹诺酮类耐药率在33.0%以下。intⅠ1的检出率为89.1%(122/137),检出dfrA17-aadA5和aar-3-aacA4两种基因盒,首次在志贺菌中检测到aar-3-aacA4基因,GenBank登录号JF271916。结论安徽省临床分离志贺菌多重耐药现象严重。临床仍可选用氟喹诺酮类和三代头孢菌素作为本地区细菌性痢疾的经验性治疗。1类整合子与志贺菌的耐药具有一定的相关性。     

嗜麦芽寡养单胞菌临床分离株对复方磺胺甲噁唑耐药机制探讨

目的了解临床分离嗜麦芽寡养单胞菌对复方磺胺甲噁唑的耐药情况，探讨其耐药机制。方法采用微量肉汤稀释法测定MⅠC．PCR法扩增Ⅰ型整合子特异的整合酶基因和sul 1基因。结果30株嗜麦芽寡养单胞菌中，5株（16．7％）表现为对复方磺胺甲噁唑高MIC，5株菌的Ⅰ型整合酶基因（intI1）和sul 1基因扩增阳性，其余均为阴性。结论嗜麦芽寡养单胞菌对复方磺胺甲噁唑的高度耐药性可能与Ⅰ型整合子的存在有关。     

59株大肠杆菌中整合子的研究

目的:研究59株耐头孢西丁大肠杆菌整合子的分类、结构及其在介导耐药中的作用。方法:利用多重聚合酶链反应(PCR)方法检测整合酶基因(intI),对其阳性菌株可变区(Int)扩增产物进行测序分析;采用微量稀释法测定22种抗生素对试验菌株的敏感性。结果:59株大肠杆菌中,45株Ⅰ类整合酶基因阳性(76%);所携带的耐药基因盒绝大多数为aadA5和dfr17;仅有2株携带β-内酰胺酶耐药基因盒;整合子阳性组抑菌浓度明显高于阴性组。结论:Ⅰ类整合子广泛地存在于耐头孢西丁大肠杆菌中;耐药基因盒是整合子阳性菌株对氨基糖苷类、磺胺类药物及氯霉素耐药的主要原因,但对介导β-内酰胺类耐药方面,不起主要作用。     

天津地区志贺菌耐药性及整合子携带调查

目的:了解天津地区志贺菌抗生素耐药性变迁及1、2类整合子携带状况。方法:K-B纸片法测定1981—1983年及2009年天津地区临床分离的57株志贺菌药敏情况。以煮沸法制备细菌总DNA作为PCR扩增模板。PCR方法扩增1、2类整合子整合酶及可变区并测序分析。PCR产物直接测序,结果经BLAST程序与GenBank数据库标准菌株比对分析。结果:1981—1983年组志贺菌对四环素、链霉素、氯霉素及复方新诺明敏感率低,3种及3种以上抗生素多重耐药率为66.67%;2009年组志贺菌对氨苄西林、链霉素、复方新诺明、哌拉西林和四环素敏感率低,3种及3种以上抗生素多重耐药率为83.33%。1981—1983年组1类整合子阳性率为87.88%(29/33),27株可变区含氨基糖苷类药物耐药基因aadA;1株宋内志贺菌可变区含甲氧苄啶耐药基因dfrA17和氨基糖苷类药物耐药基因aadA5;未发现2类整合子阳性株;2009年组1类整合酶阳性率为79.17%(19/24),可变区及3末端扩增均阴性;2类整合子阳性率87.50%(21/24),可变区含dfrA1+sat1+aadA1,介导甲氧苄啶、链丝菌素和链霉素耐药;其中17株1、2类整合酶均阳性。结论:2009年分离的志贺菌抗生素耐药较上世纪80年代分离的志贺菌增强,多重耐药菌株增加。天津地区志贺菌携带1、2类整合子。′     

儿童志贺菌Ⅰ类整合子及与产超广谱β-内酰胺酶基因关系的研究

目的：研究儿童志贺菌Ⅰ类整合子表达及其与产超广谱β-内酰胺酶（ESBLs）基因的相关性。方法：采用PCR方法检测60株产ESBLs志贺菌基因分型以及检测60株产ESBLs与60株非ESBLs志贺菌Ⅰ类整合酶基因,以分析Ⅰ类整合子与细菌耐药性的关系以及与ESBLs基因的关系。结果：儿童临床产ESBLs志贺菌耐药基因分型以CTX-M型最多见（85.0%）,其次为TEM-1型（50.0%）。产ESBL菌株基因分型分布情况以CTX-M型最多见（56.7%）,其次为TEM-1+CTX-M型（20.0%）和TEM-1型（20.0%）。产ESBLs和非产ESBLs菌株中Ⅰ类整合酶扩增阳性例数分别是55例（91.7%）和15例（25.0%）,两组整合子阳性率比较差异有统计学意义（P<0.01）,同时Ⅰ类整合子表达与ESBLs具有相关性（R=0.67,P<0.01）;Ⅰ类整合子阳性的菌株耐药性明显高于Ⅰ类整合子阴性的菌株,整合子阳性株对环丙沙星、左氧氟沙星以及复方磺胺甲噁唑耐药率较高,依次为84.3%、58.6%和90.0%。结论：儿童产ESBLs志贺菌中Ⅰ类整合子携带率明显高于ESBLs阴性菌株,显示儿童志贺菌Ⅰ类整合子与ESBLs基因之间存在密切相关性。     

阴沟肠杆菌Ⅰ类整合子可变区耐药基因的研究

目的 研究阴沟肠杆菌Ⅰ类整合子可变区基因盒的种类及位置，探讨Ⅰ类整合子携带的耐药基因盒特点，分析耐药基因盒与阴沟肠杆菌耐药性的关系。方法 采用纸片扩散法(K-B法)测定抗生素的敏感性；PCR扩增阴沟肠杆菌Ⅰ类整合子阳性株中的可变区基因盒，并测序分析。结果 可变区一共扩增出15种基因盒，其中耐药基因盒7种：aadA2-dfrA12、aadA2-aadB、aadA2、aadB、dfrA12、aac(6′)-Iica-catB3和blaIMP-4，以aadA2-dfrA12(1913bp)最常见。许多质粒DNA和染色体DNA的PCR扩增产物相同。结论 Ⅰ类整合子可变区携带的耐药基因盒以耐氨基糖昔类和耐甲氧苄氨嘧啶类抗生素基因为主。耐药基因在阴沟肠杆菌中存在水平播散和垂直传播的现象。     

大肠埃希菌中Ⅰ类整合子耐药基因的分析

目的 对临床分离大肠埃希菌株携带的Ⅰ类整合子及相关耐药基因进行筛选和分析.探讨Ⅰ类整合子在大肠埃希菌耐药中的作用.方法 对43株大肠埃希菌临床分离株做药敏试验;采用PCR扩增、DNA测序、DNA序列比对的方法 对其携带的Ⅰ类整合子相关耐约基因进行分析.结果 43株大肠埃希菌分离株对10种抗菌药物的耐药率依次为亚胺培南4.7%、阿米卡星18.6%、头孢他啶、27.9%、头孢吡肟37.2%、头孢呋辛55.8%、复方磺胺甲嗯唑58.1%、妥布霉素74.4%、庆大霉素79.1%、头孢噻肟81.4%和哌拉两林83.7%.在43株大肠埃希菌分离株中有25株含有Ⅰ类整合子,其中18株携带整合子相关耐药基因,如介导对磺胺类和氨基糖苷类约物的耐药基因等;某些菌株携带的整合子相关耐药基因相同.结论 Ⅰ类整合子在大肠埃希菌中广泛存在,整合子相关耐约基因在该菌耐药性的形成和播散中发挥作用.     

鲍曼不动杆菌Ⅰ类整合子与多重耐药相关性研究

目的 了解临床分离鲍曼不动杆菌的耐药状况、Ⅰ类整合子的分布情况,探讨Ⅰ类整合子与多重耐药的关系.方法 检测20种临床常用抗菌药物对鲍曼不动杆菌临床分离株的最低抑菌浓度(MIC).PCR扩增Ⅰ类整合酶基因.对部分Ⅰ类整合酶阳性菌株进行耐药基因盒序列分析.结果 鲍曼不动杆菌呈现多重耐药,鲍曼不动杆菌对IMP和MRP耐药率分别为0.9%和1.8%,对CPZ/SB的耐药率为35.7%,对其它抗菌药物的耐药率均大于60%,多重耐药率为76.8%(86/112),但对COL和MIN均敏感.80.4%(90/112)的菌株检测出Ⅰ类整合子.Ⅰ类整合子阳性株对多种药物的耐药率均高于阴性株,且Ⅰ类整合子阳性株多重耐药率(90%)明显高于阴性株(22.7%)(P<0.01).Ⅰ类整合子基因盒序列分析显示,Ⅰ类整合子携带aacA4,catB8和aadA13种耐药基因.结论 Ⅰ类整合子在鲍曼不动杆菌中检出率很高并与其多重耐药性关系密切.     

Possible role of integrase gene polymerase chain reaction as an epidemiological marker: study of multidrug-resistant Acinetobacter baumannii isolated from nosocomial infections

Eighty-six strains of Acinetobacter baumannii from India and Nepal were investigated for the presence of integrons in relation to multiple drug resistance by integrase gene polymerase chain reaction (PCR). Integrons were found to be present at a rate of 43.02% (37/86). Integrons were significantly correlated with multidrug resistance to several antibiotics. Class 1 integrons were detected in 81.1% of integron-positive strains, whilst 18.9% were found to be positive for class 2 integrons. The majority of class 2 integrons (71%) were encountered in strains isolated from post-operative wards of both countries. The highest integron carriage in isolates of A. baumannii (63.6%) was observed in 2005. Hence, it is likely that integrons play an important role in antibiotic resistance and possibly indicate epidemic behaviour of A. baumannii. Integrase gene PCR may be used as routine screening and identification for the surveillance of clinical isolates of A. baumannii with epidemic potential.     

Class 1 integron gene cassettes in multidrug-resistant Gram-negative bacteria in southern China

Non-duplicate multidrug-resistant Gram-negative bacteria (n=1447) isolated from January 2008 to December 2009 were investigated for the presence of integrons as well as characterisation of gene cassettes. Among 825 strains carrying the class 1 integrase gene intI1, 461 gene cassette-positive isolates were found. Thirty-eight distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP) and DNA sequencing analyses. In addition, several novel gene cassette arrays detected in this study were reported for the first time in some species: one in Escherichia coli, six in Klebsiella pneumoniae, six in Enterobacter cloacae, three in Enterobacter aerogenes, one in Proteus mirabilis, one in Acinetobacter spp., one in Stenotrophomonas maltophilia and one in Pseudomonas putida. Among them, three cassettes, including HAD-like, ΔMFS-1 and qnrVC-like genes, were originally detected in integrons.     

第一类整合子整合酶基因intI1的定位分析

目的　整合子 ( integrons)介导的细菌耐药特性已成为研究细菌耐药机制的热点 ,在研究了来自正常人携带沙门氏菌中整合子的分布和特性的基础上 ,进一步探讨整合子的基因定位。方法　从已鉴定的整合子阳性菌株出发 ,分别提取其质粒和染色体 DNA,进行质粒的接合转移试验。对染色体 DNA进行限制性酶切 ,以第一类整合酶基因 int I1( DIG标记 )为探针 ,进行 Southern杂交。结果　 4株整合酶阳性菌株不存在含有第一类整合子的接合性质粒 ,确定 4株整合子阳性菌株的整合酶基因 int I1基因位于染色体上。结论　本文发现的整合子阳性菌株对耐药基因的捕获是通过染色体 DNA介导的。     

Antibiotic resistance, integrons and Salmonella genomic island 1 among non-typhoidal Salmonella serovars in The Netherlands

The objective of this study was to investigate the antimicrobial resistance patterns, integron characteristics and gene cassettes as well as the presence of Salmonella genomic island 1 (SGI1) in non-typhoidal Salmonella (NTS) isolates from human and animal origin. Epidemiologically unrelated Dutch NTS strains (n=237) originating from food-producing animals and human cases of salmonellosis were tested for their susceptibility to 15 antimicrobial agents. Resistance to 14 of these antimicrobials, including the third-generation cephalosporins, was detected. Resistance to sulphonamides, ampicillin, tetracycline, streptomycin, trimethoprim and nalidixic acid was common (>/=10% of the strains were resistant). Resistance against three or more antimicrobials was observed in 57 isolates. The same 237 strains were studied for the prevalence of class 1 integrons, their gene cassettes and the presence of SGI1. Thirty-six isolates (15.2%) carried class 1 integrons. These integrons had ten distinct profiles based on the size of the integron and restriction fragment length polymorphism analysis. Integrons were detected for the first time in serovars Indiana and Senftenberg. Multidrug resistance was strongly associated with the presence of class 1 integrons in which the aadA2, aadA1, bla(PSE-1), dfrA1, dfrA5, dfrA14 or sat genes were present, as determined by nucleotide sequence determination. The presence of gene cassettes or combinations of gene cassettes not previously found in integrons in Salmonella was observed. SGI1 or its variants (SGI-B, -C and -F) were present in 16 isolates belonging to either serovar Typhimurium, Derby or Albany. Regardless of whether the isolate was of human or animal origin, the same resistance phenotype, integron profile and SGI1 structure could be observed.     

Class 1 integrons in environmental and clinical isolates of Pseudomonas aeruginosa

The aims of this study were to ascertain the presence and spread of class 1 integrons amongst environmental and clinical isolates of Pseudomonas aeruginosa and to characterise their variable regions. A total of 76 isolates (56 clinical and 20 environmental) were studied. The presence of plasmids was explored, and polymerase chain reaction (PCR) was used for integron detection. All amplicons were sequenced. PCR detected class 1 integrons in 26 of the 56 clinical isolates; environmental isolates were integron-free. No plasmids were found, thus all the integrons found are possibly on the chromosome. Most isolates presented one amplicon, except PA110514 and PA116136, which showed two PCR products each. Variable regions revealed that 18 strains carried only one gene involved in aminoglycoside resistance, whereas in 3 strains gene cassettes were not found. The most prevalent cassettes amongst isolates were those encoding aminoglycoside adenyltransferase B (aadB). Several of the strains had acquired the same or a highly similar cassette array as those detected in geographically distant P. aeruginosa. This finding suggests that contact with bacterial reservoirs contributes to the evolution of this pathogen towards multiresistance. Empty structures found may represent a reservoir increasing the capacity to adapt to the environment. However, these integrons are not retained when the selective pressure disappears. It is hypothesised that integrons containing gene cassettes are crucial vehicles for the rapid horizontal transfer of resistance. If this is so, reduced use of antibiotics may lead to a significant decrease in the carriage of integrons amongst P. aeruginosa strains.