1993-2000年霍乱弧菌耐药整合子的分布状况

目的检测Ⅰ类、Ⅱ类和Ⅳ类整合子及Ⅰ类整合子携带的耐药基因盒在霍乱弧菌临床菌株中的分布，分析整合子对细菌耐药性的影响。方法纸片扩散法行药物敏感试验，PCR法检测50株临床菌株Ⅰ类、Ⅱ类和Ⅳ类整合酶基因（intⅠ）阳性菌株，并对intⅠ1阳性菌株整合的耐药基因行序列分析。结果全部菌株至少对一种抗生素耐药。3株Ⅰ类整合酶基因阳性菌株的耐药基因盒PCR扩增得到1009bp的产物。序列分析表明，1009bp序列与已知的aadA 1c 100％同源，为对壮观霉素、链霉素产生耐药的基因盒；未检测到Ⅱ类整合子阳性菌株；50株临床菌株均含有第Ⅳ类整合酶基因，2株菌株第Ⅳ整合酶基因扩增得到2200bp产物，序列分析表明，在第Ⅳ类整合酶基因序列中含有一个插入片段IS1359转座酶基因。结论第Ⅰ类整合子存在于孟加拉国的临床致病弧菌中，第Ⅳ类整合子在霍乱弧菌临床菌株中分布广泛，整合子在细菌耐药中发挥作用。     

医院污水中大肠埃希菌的耐药谱及Ⅰ类整合子分析

目的调查医院污水中多重耐药菌污染情况及其耐药基因和Ⅰ类整合子携带情况。方法采集河南省郑州大学第一附属医院污水,使用16S rRNA测序和微量生化反应管鉴定细菌种属;采用PCR扩增耐药基因和Ⅰ类整合子基因盒,计算其检出率。结果根据16S测序和生化反应共分离鉴定出23株大肠埃希菌。药敏试验显示23株大肠埃希菌对四环素的耐药率为62.5%(15/23),复方新诺明为56.5%(13/23),氨苄西林为56.5%(13/23)。所有菌株均对美罗培南和亚胺培南敏感。大肠埃希菌多重耐药率为56.52%(13/23)。PCR检测耐药基因携带率aadA1基因为100%,tetA基因为95.8%,aac(6')-Ib-cr基因为79.2%,CTX基因为70.8%,tetB基因为63.5%,Qnrs基因为58.3%,SHV基因为54.2%,qepA基因为41.7%,Sul基因为35%。所有菌株均不携带KPC基因、IMP基因和NDM1基因。有21株细菌检测到Ⅰ类整合子,其中11株细菌的整合子携带空基因盒。整合子携带的大多数基因盒编码DfrA和aadA基因,有1株细菌的基因盒编码OXA-1基因。结论医院污水中的大肠埃希菌存在多药耐药现象且Ⅰ类整合子携带率高,应引起高度重视。     

多重抗药大肠杆菌中Ⅰ型整合子的分布与抗药关系

目的检测多重抗药大肠杆菌中检测Ⅰ型整合子的携带率及其对细菌抗药性的影响。方法用大肠杆菌显色．培养基分离奶牛场、猪场和肉鸡场的565株大肠杆菌,测定其对15种抗生素的MIC;用PCR扩增其中91株多重抗药性（最少耐3种抗生素）菌株的Ⅰ型整合酶基因及抗药基因盒。结果565株大肠杆菌仅对头孢噻呋、阿米卡星和硫酸粘菌素的抗药率低于50％,其余药物均表现出较高抗药率,且几乎所有细菌都对3种以上抗生素具有抗性。91株多重抗药大肠杆菌Ⅰ型整合子的平均检出率为83．52％,大多数整合aadA基因和dhfr基因,抗药性基因携带率为43．42％。结论Ⅰ型整合子对细菌多重抗药性的产生和传播起着重要作用,是临床细菌多重抗药性监测在基因水平的重要指标。     

整合子及相关基因盒在临床分离铜绿假单胞菌中的分布

目的检测Ⅰ类、Ⅱ类和Ⅲ类整合子及Ⅰ类整合子相关基因盒在铜绿假单胞菌临床分离株中的分布,分析整合子对细菌耐药性的影响。方法用纸片法对62株临床分离铜绿假单胞菌进行药敏试验;应用多重PCR法检测62株铜绿假单胞菌Ⅰ类、Ⅱ类和Ⅲ类整合子;对Ⅰ类整合子阳性菌进行整合子相关基因盒检测。结果62株菌中有40株(64.5%)含有Ⅰ类整合子,1株(1.6%)含有Ⅱ类整合子;没有检测到Ⅲ类整合子阳性菌。在Ⅰ类整合子阳性菌中,有26株携带Ⅰ类整合子相关基因盒(65.0%);分离自同一科室的部分菌株携带大小相同的基因盒;Ⅰ类整合子阳性菌株的耐药率高于整合子阴性的菌株。结论Ⅰ类整合子及整合子相关基因盒在铜绿假单胞菌临床菌株中分布广泛,整合子在细菌耐药中发挥作用。     

耐三代头孢菌素阴沟肠杆菌临床株中CTX-M型ESBLs与Ⅰ类整合子的相关性

目的研究CTX-M型超广谱β内酰胺酶（ESBLs）及Ⅰ类整合子在耐三代头孢菌素阴沟肠杆菌中的分布,进一步探讨Ⅰ类整合子与CTX-MM型ESBLs的关系.方法运用K-B法检测阴沟肠杆菌临床株的耐药表型,双纸片协同试验（DDST）初筛产ESBLs的菌株,聚合酶链反应（PCR）扩增确证产CTX-M型ESBLs和含有Ⅰ类整合子的临床菌株,套式PCR及序列测定寻找携带CTX-M型ESBLs基因盒的Ⅰ类整合子.结果37株耐药菌中,21株菌产生CTX-M型ESBLs;20株菌含有Ⅰ类整合子;在13株同时产生Ⅰ类整合子和CTX-M型ESBLs的临床株中,3株菌CH4、CH11和Q1的CTX-M型ESBLs基因盒分布在Ⅰ类整合子上.结论Ⅰ类整合子的存在增加了ESBLs在临床株中水平播散的危险,是造成多重耐药株在院内暴发流行的重要原因.     

屠宰生猪多重耐药沙门菌I类整合子与耐药基因的检测

目的了解屠宰生猪沙门菌分离株多重耐药与I类整合子及耐药基因的携带关系。方法采用K-B纸片法对屠宰生猪78株沙门菌分离菌株进行22种抗生素敏感试验;应用PCR技术对沙门菌分离菌株进行I类整合子及耐药基因检测。结果78株沙门菌分离株中有41株(52.56%)对2种以上抗生素耐药,属于多重耐药株,强力霉素-四环素-卡那霉素-氯霉素是主要多重耐药谱;41株多重耐药菌中有19株(46.34%)携带I类整合子,tetB、aph(3)-IIa和Flor基因分别检出最高。结论沙门菌多重耐药性与整合子的携带之间关系密切,耐药表型测定结果与耐药基因检测结果一致。     

儿科院内感染肺炎克雷伯菌产超广谱β-内酰胺酶基因型检测及分析

采用琼脂纸片扩散法和双纸片协同试验,对儿科院内感染肺炎克雷伯菌的耐药性及产超广谱β-内酰胺酶(ESBLs)菌株进行检测;用PCR检测耐药基因和DNA序列,分析产ESBLs菌株的基因型.结果 从422份感染标本中分离出201株肺炎克雷伯菌,产ESBLs菌株阳性率39.4%(63/201);携带blaCTX-M-11基因17株(17/63,26.9%)、blaTEM-1基因7株(7/63,11.1%)、blaSHV-2基因5株(5/63,7.8%).ESBLs阳性株对多种抗生素的耐药率高于ESBLs阴性株,只对亚胺培南敏感.提示吉林地区儿科院内感染肺炎克雷伯菌产ESBLs检出率高,并呈多重耐药性,携带三种ESBLs基因型.     

大肠埃希菌中新的耐药基因盒aadA23的变异

目的基于整合子-细菌耐药系统在细菌耐药机制中的重要作用,对成人腹泻患者的大肠埃希菌Ⅰ类整合子阳性菌株携带的耐药基因盒的基因特征进行分析.方法应用聚合酶链反应（PCR）检测Ⅰ类整合酶基因intⅠ阳性菌株并对其整合的耐药基因进行测序及用生物信息软件对序列进行分析.结果5株Ⅰ类整合酶基因阳性菌株的耐药基因盒PCR扩增得到1009 bp的产物.序列分析结果表明,1009 bp序列含有780 bp的开放阅读框,与已知的aadA23和aadA21分别有99.6%和99.5%的相似性,与aadA5只有66.4%相似,为对氨基糖苷类抗菌药物壮观霉素、链霉素产生耐药的基因盒,建议命名为aadA23b.结论随着选择环境不同,整合子整合的基因盒会发生变异,提示我们要用分子生物学的手段从基因水平分析耐药基因的遗传与变异.     

河南省人源产ESBLs鼠伤寒沙门菌单相变异株的分子特征研究

目的 分析河南省腹泻病例粪便标本中产超广谱β内酰胺酶(ESBLs) 的鼠伤寒沙门菌单相变异株耐药情况,并研究其分子学特征。方法 对河南省腹泻粪便中分离的124株鼠伤寒沙门菌单相变异株沙门菌,通过肉汤稀释法进行抗生素敏感性试验并筛选产ESBLs菌株;PCR方法检测β-内酰胺酶编码基因携带情况,采用质粒接合试验分析耐药基因的水平转移情况,应用脉冲场凝胶电泳(PFGE)进行亲缘关系分析。结果 124株鼠伤寒沙门菌单相变异株沙门菌耐药严重,其中有16株为产ESBLs菌株。16株产ESBLs菌株均携带CTX-M型耐药基因,并检测出OXA型和TEM型耐药基因;其中9株菌可将CTX-M基因通过质粒转移到大肠埃希菌J53,药敏分析发现其他抗生素的耐药性可以发生共转移。16株产ESBLs 鼠伤寒沙门菌单相变异株沙门菌经XbaⅠ酶切后共分为14种带型,无明显的优势带型。结论 检出产ESBLs 的鼠伤寒沙门菌单相变异株沙门菌基因型具有多样性,耐药基因可通过接合性质粒在不同菌属间播散。     

肉鸡源多重耐药空肠、结肠弯曲菌的耐药分子特征

目的了解肉鸡源空肠和结肠弯曲菌的耐药谱特征,检测多重耐药菌株Ⅰ类整合子/基因盒、gyrA基因突变位点、tetO基因、23S rRNA突变位点的分子特征。方法利用PCR检测弯曲菌Ⅰ类整合子/基因盒的存在情况;利用MAMA PCR技术检测弯曲菌gyrA基因第257位碱基的突变情况;针对弯曲菌23S rRNA的V区2075突变位点检测突变菌株。结果多重耐药菌株占分离株的94.5%。146株多重耐药空肠和结肠弯曲菌中Ⅰ类整合子检出率为98.6%,有78株菌株检出3种基因盒,1 000 bp+750 bp+500 bp+250 bp为主要谱型,所占比例为92.3%;有131株在gyrA喹诺酮类耐药决定区发生突变,突变率为92.9%。127株四环素耐药弯曲菌tetO基因的检出率为95.3%。81株红霉素耐药菌株中,23S rRNA的V区2075处突变发生率为96.3%。结论空肠和结肠弯曲菌分离株携带aadA2耐药基因盒,与氨基糖苷类药物的耐药性相关;gyrA基因突变、tetO基因的携带以及23S rRNA突变,与弯曲菌对喹诺酮、四环素和大环内酯类耐药密切相关。     

Class 1 integrons and resistance gene cassettes among multidrug resistant Salmonella serovars isolated from slaughter animals and foods of animal origin in Ethiopia

The present study was undertaken to identify and characterize integrons and integrated resistance gene cassettes among multidrug resistant (MDR) Salmonella isolates from slaughter animals and food products of animal origin in Ethiopia. A total of 98 epidemiologically unrelated Salmonella isolates comprising 13 serovars were characterized using serotyping, phage typing, antimicrobial resistance testing and the pulsed-field gel electrophoresis (PFGE) method. Integron-PCR was used to detect the presence of class 1 and class 2 integrons in the MDR strains. The associated individual resistance gene cassettes were identified using specific PCRs and DNA sequencing. The location of the integrons was determined by Southern blot hybridization analysis. Among the Salmonella serovars, a high level of antimicrobial resistance was found to streptomycin (82.6%), tetracycline (75.5%), sulfamethoxazole (60.2%), spectinomycin (53.1%), ampicillin (42.8%), nalidixic acid (34.7%), nitrofurantoin (30.6%), trimethoprim (27.5%), gentamicin (20.4%) and ciprofloxacin (19.4%). Class 1 integrons were detected in 53.1% of the MDR isolates comprising serovars Anatum, Braenderup, Kentucky, Saintpaul and Typhimurium. Of the class 1 integron positive isolates 61.5% harboured the integron-associated gene cassettes: aadA2, aadA2+bla(PSE-1), dfrA1-aadA1 and dfrA12-orf-aadA2 (amplicon sizes 1000 bp, 1000+1200 bp, 1600 bp and 1900 bp, respectively). The chromosomally located aadA2 and aadA2+bla(PSE-1) resistance gene cassettes occurred exclusively in S. Typhimurium DT104 isolates, the other cassettes were found on large plasmids in several serovars. An aacCA5-aadA7 gene cassette array (amplicon size 1600 bp) was exclusively found in all MDR S. Kentucky strains of R type Str/SpeSmxGenNalAmpTetCipCef and this integron was shown to be chromosomally located. Results of the present study indicate that class 1 integrons carrying gene cassettes, which confer resistance to different classes of antimicrobials such as aminoglycosides, beta-lactams and trimethoprim are widespread among the MDR Salmonella serovars isolated from slaughter animals and food products of animal origin in Ethiopia indicating the important role of these genetic elements in the dissemination of multidrug resistance.     

Characterization of class 1 integrons and antimicrobial resistance in CTX-M-3-producing Serratia marcescens isolates from southern Taiwan

The objective of this study was to characterize the gene cassettes of class 1 integrons and antimicrobial resistance among CTX-M-3-producing Serratia marcescens isolates from different specimens in southern Taiwan. One hundred and twenty-two isolates (70.5%) of 173 CTX-M-3-producing S. marcescens isolates were positive for class 1 integrons, including 53.3% of blood isolates, 94.1% of urine isolates, and 87.2% of sputum isolates. No class 2 or class 3 integrons were detected in this study. By PCR with primers 5'-CS and 3'-CS for the amplification of gene cassettes regions, amplicons ranging from 0.7 to 3.0 kb in length were found in 108 (88.5%) of the 122 class 1 integron-containing isolates of CTX-M-3-producing S. marcescens isolates. Ten different types by pattern of amplicons for class 1 integrons were obtained. The Type I amplicon (46.3%) harbors two different class 1 integrons containing the gene cassette of aadA2 and aadB-catB3, respectively, and was most prevalent in the gene cassette region-positive S. marcescens isolates, followed by the Type II amplicon, which harbors one class 1 integron containing the gene cassette dfrA12-orfF-aadA2 (28.7%). Most of the S. marcescens isolates (66.7%, 8/12) harboring three different class 1 integrons (Type IV amplicon) were found in blood isolates. Class 1 integrons were conjugally transferred to recipients in 92.0% of S. marcescens harboring two different class 1 integrons containing the gene cassettes aadA2 and aadB-catB3, respectively. The transfer rate of class 1 integron carrying dfrA12-orfF-aadA2 was detected in 77.4% of S. marcescens isolates. The results showed that all those isolates with conjugative transfer of integrons carried their class 1 integrons on the conjugative plasmids.     

Variable gene cassette patterns of class 1 integron-associated drug-resistant Escherichia coli in Taiwan

This study characterized class 1 integrons in Escherichia coli in Taiwan. The stability and changes in gene cassettes inserted into integrons were also evaluated. The study included 436 clinical strains of E. coli isolated in 2002. Class 1 integrons were characterized by polymerase chain reaction and direct sequencing. Genetic localization of class 1 integrons was determined by conjugal transfer and Southern hybridization. The results indicated that 64% of E. coli isolates carried class 1 integrons. Molecular analysis revealed that the class 1 integrons harbored 13 different antimicrobial resistance gene cassettes and two unknown gene cassettes; the predominant cassettes were aadA and dfrA. Novel gene cassettes first recovered from E. coli were aacA4 and linF. Cassette arrays orfD-aacA4-catB8 and aadA1-linF were also observed. Gene cassette dfrA12-orfF-aadA2 was stable. The class 1 integron and dfrA17-aadA5 gene cassette were located on the same transferable plasmids and were capable of transmission. Therefore, the increased drug resistance of clinical isolates may be explained by antibiotic selective pressure and widespread presence of integrons. Under antibiotic selective pressure, gene cassette-mediated resistance may not be easily lost. The potential role of integrons in the uptake and dissemination of resistance genes by plasmid between species of bacteria may decrease the therapeutic effectiveness of antibiotics.     

Molecular diversity of class 1 integrons in human isolates of Aeromonas spp. from southern Taiwan

This work studies antimicrobial resistance and class 1 integrons of Aeromonas spp. in human isolates from southern Taiwan. PCR amplification and DNA sequence analyses were performed to characterize the gene cassette regions of the class 1 integron in 204 isolates of Aeromonas hydrophila, 36 isolates of A. sobria, 23 isolates of A. veronii, and 4 isolates of A. caviae. By using Southern hybridization with an intI1 probe to determine the presence of class 1 integrons in the 9 isolates of Aeromonas spp. harboring plasmid DNA, only 2 isolates, one A. veronii AV69 harboring 176-kb plasmid DNA, and one A. hydrophila AH207 harboring 149-kb plasmid DNA were identified. A conjugation experiment was carried out with 2 isolates of A. veronii AV69 and A. hydrophila AH207. Only one transconjugant of Escherichia coli AH207, containing 149-kb plasmids obtained from A. hydrophila AH207, was identified. ERIC-PCR analysis was performed to analyze the genetic relatedness in all isolates of Aeromonas spp. that carry class 1 integrons. The results of cluster analysis in this experiment revealed that none of these isolates were clonal, which may indicate that they were not related to the outbreak. Among the 267 isolates tested, class 1 integrons were detected in 37 isolates (13.9%) of Aeromonas spp. from humans. No class 2 or class 3 integrons were detected in this study. Gene cassette structures were identified in 30 (81.0%) of 37 isolates of Aeromonas spp. containing class 1 integrons. The gene cassette of dfrA12-orfF-aadA2 was the most prevalent in the gene cassette array (16.0%), followed by arr3-aacA4 (13.3%) and dfr2d-catB3-aadA1 (10.0%). Four novel arrays of gene cassettes were also identified, namely, dfr2d-catB3-aadA1, aadA1-aac(6')-II, aadA4a, and aac(6')-II-blaOXA-21-catB3. This is the first report of Aeromonas spp. isolates from humans.     

临床痢疾志贺菌整合子检测及耐药基因盒序列分析

目的 检测印度东部1988、1995和2002年部分临床分离痢疾志贺菌中细菌耐药关系密切的1、2、3类整合酶基因及整合子携带的耐药基因盒的分布,分析整合子系统对志贺菌耐药的影响.方法 纸片扩散法检测实验菌株对药物的敏感性;应用PCR方法对16株临床耐药菌株进行1、2、3类整合酶基因(intI)筛选,对阳性样本可变区基因盒序列进行鉴定分析.结果 所有16株菌均耐4种及4种以上药物,包括β-内酰胺类、氨基糖苷类、四环素类、磺胺类、氯霉素类和喹诺酮类.13株菌检出1类整合酶基因,全部菌株含2类整合酶基因,即发现同时存在两种整合子结构菌株,未检测到3类整合酶基因.1类整合酶插入基因盒以blaara30-aadAl基因家族为主,分别对β-内酰胺类抗生素、链霉素、壮观霉素耐药;2类整合酶插人基因盒以dfrAl-satl组合为主,分别对甲氧苄氨嘧啶、链丝菌素耐药,同时在4株菌中发现dfrAl-satl-aadAl基因盒组合.结论 2类整合子普遍存在于临床志贺菌中.整合子与志贺菌的多重耐药具有密切相关性.     

First detection and sequence analysis of the bla-CTX-M-15 gene in Lebanese isolates of extended-spectrum-beta-lactamase-producing Shigella sonnei

The emergence in Shigella species of extended-spectrum beta-lactamases (ESBL) that impart resistance to third-generation cephalosporins is a growing concern world-wide. So far, however, ESBL-producing Shigella have only been reported seven times, albeit from seven different countries. In Lebanon, three ESBL-producing clinical isolates of S. sonnei were recovered from 30 cases of shigellosis diagnosed between July 2004 and October 2005. All three were found to be resistant to amoxycillin, cefotaxime, ceftazidime, aztreonam, trimethoprim/sulphamethoxazole, gentamicin, and kanamycin. Each harboured the bla-CTX-M gene, and the results of sequence analysis indicated this to be of the bla-CTX-M-15 type and encoded on a 70-kb plasmid, flanked by an insertion element (ISEcp1). The bla-TEM-1 gene was also detected on the chromosomes of two of the ESBL-producing isolates. Class-2 integrons containing dhfr1, aadA1 and sat1 genes were detected on the chromosomes of all three isolates but not on the plasmids. Fluoroquinolone-modifying factors [QnrA, QnrB, QnrS or AAC(6')-Ib-cr] were not detected. The results of RAPD analysis, combined with data on antimicrobial susceptibility, indicated that each isolate was unique. In conclusion, the emergence of ESBL-producing isolates of S. sonnei has been demonstrated for the first time in Lebanon. The resistance of these isolates to third-generation cephalosporins was mediated by the CTX-M-15 enzyme, which was plasmid-encoded.     

Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt

Up to this date, nothing is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals in Africa. Therefore, this study was carried out to screen the incidence of multidrug-resistant (MDR) strains of Salmonella from neonatal calf diarrhea in Egypt and also to characterize the molecular basis of this resistance. Nine unique Salmonella isolates were obtained from 220 fecal samples, and six of these showed multidrug resistance phenotypes and harbored at least two antimicrobial resistance genes. Four were Salmonellaenterica serovar Typhimurium and two were S.enterica serovar Enteritidis. Class 1 integrons were identified in all MDR Salmonella isolates. The identified gene cassettes within class 1 integrons were as follows; aminoglycoside adenyltransferase type A (aadA1, aadA2 and aadA5), which confer resistance to streptomycin and spectinomycin, and dihydrofolate reductase gene cassettes (dfrA1, dfrA15 and dfrA15), which confer resistance to trimethoprim. A class 2 integron containing dfrA1-sat2-aadA1 gene cassettes was identified in only one isolate of S. enterica serovar Enteritidis. The β-lactamase-encoding gene, bla_TEM-1, was identified in five isolates and the extended-spectrum β-lactamase-encoding genes, bla_CMY-2 and bla_SHV-12, were identified in S. enterica serovar Typhimurium. Furthermore, the plasmid-mediated quinolone resistance genes, qnrB, qnrS and aac(6′)-Ib-cr, were also identified. To the best of our knowledge, this is the first report of qnrS in S. enterica serovar Enteritidis, qnrB in S. enterica serovar Typhimurium, and aac(6′)-Ib-cr in Salmonella of animal origin. Also, this is the first report of the molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Africa.