<i>Escherichia coli</i>Harbouring Resistance Genes, Virulence Genes and Integron 1 Isolated from Athi River in Kenya

Rivers can act as reservoirs of highly resistant strains and facilitate the dissemination of resistance, virulence and integron 1 genes. A cross-sectional study was carried out where 318 water samples were collected (53 from each site) and from the samples, 318 E. coli isolates were analysed for resistance genes, virulence genes and integron 1 using Polymerase Chain Reaction. 22% of the isolates had blaTEM, 33% had blaCTX-M and 28% had blaCMY. Prevalence of typical Enteropathogenic E. coli strains (carrying both eae and bfp genes) was 5% while the prevalence of atypical Enteropathogenic E. coli (carying only eae) was 1.8%. The prevalence of Enteroaggregative E. coli carrying the aggr genes was 11%. The prevalence of Enterotoxigenic E. coli encoding only lt toxin was 16 (5%) and while those carrying only st toxin was 6.9%. The prevalence of Enteroinvasive E. coli strains encoding as IpaH was 5% while that of strains, adherent invasive E. coli, carrying adherent invasive gene inv was 8.7%. 36% isolates were positive for class 1 integrons which were mostly isolated near the sewage effluent from waste treatment plant. Anthropogenic activities and close proximity to sewage treatment plant were found to play a key role in pollution of water body and accumulation of resistance and virulence genes. These results suggest that waste treatment plant may act as reservoir of resistance, virulence and integron 1 genes and is a potential risk to human and animal health in the region.

Detection of class 1 integron in Acinetobacter baumannii isolates collected from nine hospitals in Turkey

Objective:To investigate the antibiotic resistance genes inserted into class I and class 2integrons in Acinetobacler baumannii[A.baumannii)isolates obtained from nine different cities in Turkey.Methods:A collection of 281 A.baumannii clinical isolates were collected from nine diferenl state hospitals in Turkey and were confirmed as A.baumannU by conventional biochemical,API testing and bla_(-OXA-51)specific PCR.The isolates were examined by PCR for existence of class I and2 integron gene cassettes.Results:They were characterized by antimicrobial susceptibility testing and the highest resistance rates were determined for piperacillin(90.03%),ciprofloxacin(87.54%),cefepime and trimethoprim/sulfamethoxazole(81.13%).The lowest resistance rates was for cefotaxime(3.55%).class 1 integrons were detected in 6.4%(18/281)of A.baumannii strains and no class 2 integron was detected.The gene casselles of class 1 inlegrons AacCI-AAC(3)l-aadAI,AacCI-aadA1,AAC(3)-I,AAC(3)-I-AAC(3)-I-aadA1,TEM-1.AAC(3)-I-aadA1-AAC(3)-I-AAC(3)-I,AAC(3)-I-AAC(3)-I-AAC(3)-I-aadA1,AAC(3)-I-aadA1,AAC(3)-I-AAC(3)-I,AAC(3)-I-aadA1-AAC(3)-I-aadA1,AAC(3)-I-AAC(3)-I-aadA1-AAC(3)-I-aadA1 were detected in eighteen strains.The aac genes family were most frequently found integrated into the class 1 inlegrons and it was followed by aadA genes and TEM-1 genes.Conclusions:This is an extensive study on the distribulion of class 1 integron among A.baumannii in Turkey.In addition to these,two new alleles were observed.Their percentage rates of similarity to other cassettes are 95%aadA1(TK A18)and 89%,aadA 1(ANKA3).

Characterization of Class 1 Integron Gene Cassettes among Clinical Bacteria Isolated from One Large Hospital in Northern China

The class 1 integron and complex gene cassettes among different species of clinical isolates in northern China were characterized in this study. 383 clinical isolates were obtained from northern China, and class 1 integrons containing gene cassettes widely distributed among gram negative clinical isolates was observed. We find that the class 1 integron showed positive correlation with multidrug resistance phenotype of gram negative bacteria. In addition, we find that isolates belonged to one species harbored different types of gene cassette arrays, while same types of gene cassette arrays were observed in different species of isolates. The diversity of gene cassette arrays among the isolates indicated the complexity of multidrug resistance in clinical isolates in northern China.

Screening of Class 1 and Class 2 Integrons in Clinical Isolates of Pseudomonas aeruginosa Collected from Seven Hospitals in Turkey:A Multicenter Study

Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide, and their infections are difficult to treat due to acquired resistance to many antibiotics. This study aimed to detect class 1 and 2 integrons and antibiotic susceptibility of clinical isolates of P. aeruginosa. Two hundred and five P. aeruginosa strains were collected from the seven general state hospitals in Turkey. They were characterized by antimicrobial susceptibility testing, screened for class 1 and class 2 integrons, and evaluated for the association between antibiotic resistance phenotypes and the presence of integrons. intI gene was amplified in 10 isolates (4.87%) by PCR and in seven isolates of them (70%) were found different gene cassettes. The aadA gene integrated into the class 1 integrons was most frequently found and it was followed by aac genes and blaOXA family genes. Sequence analysis of variable regions of the class 1 integrons showed five gene cassette arrays;aadA1(99%), aac(3)-Id(82%)-orf-aac(3”)-Ia(99%), aac(3)-Ie(83%)-blaoxa10(100%)- aadA1 (100%), aadA6(99%, 100%), aac(6’)-I(97%)-orf-aadA2(99%). No class 2 integron was detected. This study is the first multicenter study for class 1 integrons and it indicates the low rate of presence of class 1 gene cassette in P. aeruginosa.

Correlation of the antiseptic resistance in Acinetobacter baumannii and the antiseptic resistance gene qacEΔ1 located in classⅠintegron

In the past decade, uses of antiseptics and disinfectants in hospitals and other health care centers are rather common, but the chance to develop resistance to antiseptics and disinfectants is also increased. Acinetobacter baumannii is one of the opportunistic bacteria involving in the nosocomial infection. In the present study, the correlation of the antiseptic resistance in A. baumannii and the antiseptic resistance gene qacEΔ 1 was investigated by means of determination of MICs. Meanwhile, the MICs of glutaraldehyde, chlorhexidine, benzalkonium bromide, iodophor and trichloroisocyanurate to 80 clinical isolates of A. baumannii were detected by tube dilution assay and the resistance genes intll and qacE Δ 1 in these isolates were amplified by PCR and verified by DNA sequencer. It was found that the MIC50 for these 5 antiseptics tested were 32, 8, 8, 4 and 1 μg/ml respectively, and the detection rates of intll and qacEΔ1 gene were 60.0% and 77.6% respectively. In addition, 55% of the 80 isolates simultaneously possessed both intll and qacEΔ1 gene, and the percentage of antiseptic resistance of A. baumannii carring both genes to benzalkonium bromide were higher than that without these two genes, however, there was no significant difference between intll and qacEΔ1 gene. The result in bactericidal efficiency assay indicated that chlorhexidine could still produce rapid and strong bactericidal effect at concentration of 1 MIC after 10 rain exposure. These results suggest that the antiseptic resistance of A. baumannil to various antiseptics is correlated with the presence of the antiseptic resistance genes qacEΔ 1 in bacteria, thus warning that the increase of the antiseptic resistance should not be ignored and the relative high concentration or prolonged application time is required to achieve a sufficient bactericidal effect.

基因捕获元件int1和ISCR1在临床菌株中的分布及与细菌耐药性的关系研究

目的研究临床多种细菌中的Ⅰ型整合酶编码基因int1和ISCR1的分布情况,探究其与细菌多药耐药表型的关系。方法收集2011-2012年河北省某医院临床菌株,应用VITEK2COMPACT全自动微生物生化鉴定系统和16SrDNA序列分析进行种属鉴定,利用PCR方法进行int1基因和ISCR1筛查;两种可移动元件的携带情况和菌株的多药耐药表型之间的关系采用卡方检验进行统计计算。结果共收集临床菌株372株,包括325株革兰氏阴性菌和47株革兰氏阳性菌;int1基因和ISCR1在22种(属)175株和18种(属)90株革兰氏阴性细菌中检出,同时在17种(属)71株革兰氏阴性细菌中检出,革兰氏阳性细菌int1基因和ISCR1检测均为阴性;通过对数据分层对比,统计分析发现,在int1基因不存在时,ISCR1的存在与否与菌株是否为多药耐药的关系明显,差异有统计学意义(P<0.01),其存在亦能介导更广泛种类药物耐受,差异亦有统计学意义(P<0.01)。在int1基因存在时,ISCR1的存在能介导更广泛种类药物耐受,差异有统计学意义(P=0.02);在ISCR1不存在时,int1基因的存在与否与菌株是否多药耐药的关系明显,差异有统计学意义(P<0.01),其存在亦能介导更广泛种类药物耐受,差异亦具有统计学意义(P<0.01)。而在ISCR1存在的情况下,上述两种关系不明显。结论基因捕获元件int1基因及ISCR1在革兰氏阴性临床菌株中分布广泛,并与细菌的多重耐药、泛耐药明显相关,特别是ISCR1;应加强可移动元件int1基因或ISCR1流行状况的监测,为其在耐药基因捕获中的作用机制研究和控制多药耐药细菌在临床散播提供可靠的基础数据。

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。

两株阴沟肠杆菌的DHA-1型耐药基因的转移方式探讨

目的 通过确定编码AmpC酶DHA 1型基因是存在于整合子还是质粒上 ,来探讨此类AmpC酶的播散方式。方法 分别提取细菌的染色体DNA和质粒DNA通过斑点杂交的方式 ,并将整合子聚合酶链反应(PCR)产物测序 ,来确定编码AmpC酶DHA 1型基因存在的位置。 结果 2株阴沟肠杆菌的斑点杂交试验均在质粒DNA中发现AmpC酶DHA 1型基因 ,而染色体DNA和整合子片断中未找到相关基因。 结论 这 2株阴沟肠杆菌的AmpC酶DHA 1型的基因均存在于质粒上 ,说明他们的AmpC酶是通过质粒的转移而散播的。

铜绿假单胞菌1型整合子与多重耐药性关系

目的了解铜绿假单胞菌1型整合子携带情况及耐药性,探讨其与多重耐药的关系,为烧伤患者抗生素选择提供依据。方法采用PCR技术对26株分离于烧伤病房的铜绿假单胞菌进行1型整合子检测;用K-B纸片法检测细菌对13种抗菌药物的耐药谱;分析1型整合子与铜绿假单胞菌多重耐药的关系。结果26株铜绿假单胞菌检出携带1型整合子16(61.5%);整合子阳性菌株对13种抗生素耐药率由高到低依次为妥布霉素、氧氟沙星和庆大霉素(93.8%)、环丙沙星(87.5%)、头孢曲松和头孢噻肟(62.5%)、阿米卡星(56.2%)、安典南(50.0%)、亚胺培南和头孢哌酮(43.8%)、头孢吡肟、哌拉西林和头孢他啶(37.5%)。26株铜绿假单胞菌的多重耐药率(耐4种以上抗生素)为61.5%(16/26),其中1型整合子阳性菌株多重耐药率占93.8%(15/16),阴性菌株多重耐率为10%(1/10),1型整合子阳性菌株多重耐药率明显高于阴性株(P<0.01)。结论铜绿假单胞菌1型整合子阳性携带率较高,并与多重耐药有密切关系。

污水中大肠埃希菌携带第1类整合子的鉴定和特征分析

目的:探讨污水中大肠埃希菌携带第1类耐药整合子的分布、特征及所含基因盒的种类。方法:常规方法分离大肠埃希菌;纸片扩散法对9种抗生素进行耐药性监测和分析;PCR鉴定1类整合子阳性株;PCR产物测序并对结果进行分析。结果:42份标本中分离出24株大肠埃希菌,为多重耐药菌株,耐药谱为氨苄西林-复方磺胺-红霉素-四环素-链霉素-庆大霉素。有3株(12.5%)鉴定出1.6 kb 1类整合子,PCR产物测序得知携带pse-1-aadA1、pse-1-aadA2、pse-1-aacA1基因盒,传递对β-内酰胺类-氨基糖苷类抗生素的耐药性。结论:1类整合子存在于污水来源的大肠埃希菌中,并携带相应的耐药基因盒,决定着对β-内酰胺类-氨基糖苷类抗生素的耐药性。

食品中变形杆菌第1类整合子鉴定和特征

目的探讨肉类食品中变形杆菌第1类整合子的分布、特征及所含基因盒的种类。方法常规方法分离变形杆菌;纸片扩散法对9种抗生素进行耐药性监测和分析;PCR鉴定1类整合子;阳性株PCR产物测序并对结果进行分析。结果91份标本中共鉴定出27株变形杆菌,有13株(48.1%)对至少4种以上抗生素表现多重耐药,耐药谱为复合磺胺-氨苄西林-红霉素-四环素-链霉素。13株中有3株鉴定出1类整合子,2株携带0.75和1.8 kb 2个整合子。1株携带1.0 kb整合子,PCR产物测序,1.0 kb整合子携带aadA2基因盒,传递对氨基糖苷类抗生素的耐药性。结论肉类食品中变形杆菌携带第1类整合子,1.8 kb整合子携带pse-1-aadA1-dfrA14基金盒,传递对β-内酰胺类、磺胺类、氨基糖苷类抗生素的耐药性。携带复杂的基因盒,决定着对β-内酰胺类、氨基糖苷类和磺胺类抗生素的耐药性。

临床分离多重耐药鲍曼不动杆菌和铜绿假单胞菌Ⅰ类整合子与ISCR1研究

目的了解临床分离多重耐药鲍曼不动杆菌和铜绿假单保菌Ⅰ类整合子与插入序列共同区1(ISCR1)的分布情况。方法 2010年1月至2013年12月共收集我院126株多重耐药鲍曼不动杆菌和89株多重耐药铜绿假单胞菌,采用PCR法扩增Ⅰ类整合酶、Ⅰ类整合子基因盒、ISCR1、ISCR1可变区。Ⅰ类整合基因盒和ISCR1可变区的PCR产物分别用HinfⅠ、RasⅠ进行酶切,相同类型酶切图谱的PCR产物随机挑取一例进行测序,测序结果在Gen Bank中用Blast N进行核酸序列同源性搜索。结果在多重耐药鲍曼不动杆菌中,检出79例Ⅰ类整合酶,67例Ⅰ类整合子基因盒阳性,含有6种类型;59例ISCR1阳性,其中9例ISCR1可变区阳性。在89株多重耐药铜绿中,检出41例Ⅰ类整合酶,36例Ⅰ类整合子基因盒阳性,含有10种类型;9例ISCR1阳性,其中4例ISCR1可变区阳性。有8株鲍曼不动杆菌和2株铜绿假单胞菌同时携带Ⅰ类整合子和ISCR1可变区结构。结论在多重耐药铜绿假单胞菌和鲍曼不动杆菌中,Ⅰ类整合子和ISCR1分布较广。

铜绿假单胞菌群体感应系统分子受体基因lasR与Ⅰ类整合酶intI1基因表达的相关性分析

目的通过研究铜绿假单胞菌Ⅰ类整合子阳性株群体感应系统基因lasR与Ⅰ类整合子整合酶基因intI1表达水平的相关性,初步探讨群体感应系统对Ⅰ类整合子的调控机制。方法通过荧光定量RT-PCR方法检测铜绿假单胞菌群体QS信号分子受体基因lasR和Ⅰ类整合子整合酶基因intI1在液相菌与生物被膜菌中mRNA的表达水平,分析两基因表达的相关性。结果铜绿假单胞菌在生物被膜中群体感应系统lasR基因及Ⅰ类整合子intI1基因的mRNA表达水平高于液相菌,两者表达水平呈现正相关性(r=0.695,P<0.05)。结论铜绿假单胞菌群lasR和intI1基因表达呈正相关,提示intI1基因表达可能与群体感应系统的调控作用有关。

Prevalence of class 1 integron and its gene cassettes carrying antibiotic resistance genes in drinking water treatment and distribution systems

Class 1 integrons are vital mobile genetic elements involved in the environmental transmission of antibiotic resistance genes(ARGs).However,knowledge about the diversity and abundance of class 1 integrons and gene cassettes during drinking water treatment and distribution is still limited.In this study,we aimed to uncover the prevalence of class 1 integrons in the drinking water treatment and distribution systems with the combination of culture-dependent and culture-independent methods.Further,we applied the nanopore sequencing method to characterize the diversity and arrangement of ARGs carried by class 1 integron-associated gene cassettes.A total of 42 isolates were inti-positive among the 208 strains isolated from drinking water,which tended to confer multi-drug resistance compared with intll-negative isolates.The absolute abundance of the intl1 average i.15×10^(9) copies/L in the source water and underwent the most significant reduction of over 99.9%after liquid chiorine disinfection.Furthermore,nanopore sequencing revealed that the class 1 integron-associated gene cassettes carried 51 subtypes of ARGs in drinking water,mainly conferring resistance to aminoglycosides and trimethoprim.The treatment processes,especially liquid chlorine disinfection,reduced most of the ARGs carried by gene cassettes,though some of the ARG subtypes persisted along the treatment and distribution ike aac(6)-Il,aadA,and dfrB2.The antibiotic resistance gene cassette array laac(6')-Ⅱ arr was most frequently detected,especially in the chlorinated water.This study underlined that drinking water was potential reservoir for integron-mediated ARGs transfer,indicating that the health risks of resistance gene cassettes in class 1 integrons deserved urgent attention.

临床分离产IMP-1型金属酶非发酵菌的耐药机制研究

从临床收集耐亚胺培南的铜绿假单胞菌和鲍曼不动杆菌共50株,进行头孢他啶和2-巯基乙醇的表型协同试验(CAZ+ME),然后进行金属酶IMP-1基因的PCR检测。选取IMP-1阳性株测序,用PCR方法检测有无一类整合子基因(IntI1)。表型的检测发现有28株为协同阳性,其中铜绿假单胞菌27株,鲍曼不动杆菌1株。PCR和测序检测出其中一株铜绿假单胞菌含有IMP-1基因,同时也含有IntI1基因。首次在中国西部地区发现产IMP-1型金属酶、同时也含有一类整合子的铜绿假单胞菌,对于临床上研究细菌的耐药性传播具有重要意义。

肺炎克雷伯菌第1类整合子的鉴定和耐药性分析

目的了解来源于不同临床标本的肺炎克雷伯菌的耐药性、携带第1类耐药整合子的特征及基因盒的种类．方法使用常规方法分离肺炎克雷伯菌；运用法国梅里埃VITEK细菌分析系统进行鉴定；应用纸片扩散法对15种抗生素进行耐药性监测和分析；应用PCR法扩增第1类整合子；对PCR产物纯化、测序，并对结果进行分析．结果来源于不同临床标本的64株肺炎克雷伯菌100％耐药，32株（50％）表现多重耐药，9株耐4种抗生素，耐药谱为氨苄西林．链霉素．复方新诺明-四环素；20株（31．2％）为第1类整合子阳性株，其中17株整合子大小为1．0kb，2株为1．6kb，1株含有1．0kb和1．6kb两个整合子；PCR产物测序显示1．0kb整合子携带pse-1-aadA1基因盒，1．6kb整合子插入了pse-1-aadA2，pse-1-aacA1基因盒，双重整合子株插入了pse-1-aadA1和pse-1-aacA1基因盒．结论第1类整合子存在于各种临床标本分离的肺炎克雷伯菌中，且与多重耐药性相关，主要决定着对β内酰胺类和氨基糖苷类抗生素的耐药性．

产志贺毒素大肠埃希菌中国分离株第1类整合子-基因盒的特征

目的:探讨产志贺毒素大肠埃希菌中国分离株携带第1类整合子的特征及所含基因盒的种类。方法:采用纸片扩散法对8株产志贺毒素大肠埃希菌中国分离株进行抗生素耐药性监测;制备细菌转接合子,采用PCR技术鉴定第1类整合子,并对PCR产物测序及结果分析。结果:4株产志贺毒素大肠埃希菌对复合磺胺、四环素、氨苄西林、环丙沙星表现多重耐药,其中2株同时对链霉素和壮观霉素耐药。PCR检测第1类整合子1000 bp 2株,PCR产物测序1000 bp整合子携带腺苷酰基转移酶(aadA1)基因盒并存在于7800 bp可接合质粒上,与sul 1和qacEΔ1基因连锁,分别传递着对链霉素、壮观霉素、复合磺胺和季胺盐类消毒剂的耐药性。结论:产志贺毒素大肠埃希菌中国分离株可接合质粒上第1类整合子携带的aadA1耐药基因盒是该菌株对氨基糖苷类抗生素耐药产生和扩散的重要原因。

食品中大肠埃希菌中耐药1型整合子检测及多态性分析

目的:了解食品中大肠埃希菌中耐药1型整合子的存在频率,并分析1型整合子可变区DNA碱基大小,以探讨食品中大肠埃希菌耐药基因的携带情况和多态性。方法:普通PCR检测50株分离于食品的大肠埃希菌1型整合子,并采用长片段PCR方法扩增1型整合子可变区序列,计算可变区DNA碱基长度。结果:50份样本有17份检出1型整合子结构(34%),长片段PCR扩增1型整合子DNA平均长度为2321 bp,根据可变区分子量大小判断,共有5种整合子结构。结论:食品中大肠埃希菌携带1型整合子阳性较普遍,并具有一定的多态性。

健康学生大肠埃希菌第1类整合子的鉴定及特征

目的:探讨健康学生肠道大肠埃希菌携带第1类整合子的分布及所含基因盒的特征。方法:常规方法分离大肠埃希菌;纸片扩散法对10种抗生素进行耐药性监测和分析;PCR鉴定第1类整合子阳性株;PCR产物测序并对结果进行分析。结果:从97份标本中鉴定出大肠埃希菌76株,其中25株(32.8%)为多重耐药,耐药谱为复合磺胺-氨苄西林-四环素-红霉素-链霉素。25株中有14株(56.0%)鉴定出1类整合子,1 800 bp10株,750 bp 4株。PCR产物测序得知1 800 bp整合子携带aadA1-dfrA14-orf基因盒,传递对氨基糖苷类-磺胺类抗菌药物的耐药性;750 bp整合子携带dfrA14基因盒,传递对磺胺类抗菌药物的耐药性。结论:不同种类的1类整合子存在于健康人大肠埃希菌中,携带耐药基因盒,决定多重耐药性。

肉鸡屠宰场多重耐药沙门氏菌Ⅰ类整合子与磺胺类耐药基因(sul1、sul2和sul3)的检测

目的:了解肉鸡屠宰场多重耐药沙门氏菌Ⅰ类整合子及磺胺类耐药基因携带情况。方法:采用纸片扩散法对肉鸡屠宰场84株沙门氏菌分离株进行10种抗生素敏感性实验;应用聚合酶链式反应(polymerase chain reaction,PCR)方法检测多重耐药沙门氏菌sul1、sul2、sul3和int1基因。结果:84株沙门氏菌对氨苄西林和萘啶酸耐药率高达100%,对环丙沙星、四环素、甲氧苄啶/磺胺甲噁唑、大观霉素、氟苯尼考、庆大霉素耐药率分别为39.29%、35.71%、35.71%、35.71%、22.62%、14.29%。38株沙门氏菌对3种及以上抗生素耐药,属于多重耐药菌株。38株多重耐药菌株中,有20株携带Ⅰ类整合子。30株对甲氧苄啶/磺胺甲噁唑耐药的菌株中,均能检测到sul1或sul2或sul3基因,与表型耐药100%符合,其检出率分别为40%、100%、63.3%。结论:肉鸡屠宰场中沙门氏菌耐药现象已不容乐观,沙门氏菌多重耐药性与Ⅰ类整合子的携带之间关系密切,且Ⅰ类整合子在磺胺类耐药菌株的产生中起到重要作用。