目的:分析肺炎克雷伯菌8种毒力基因在肺炎克雷伯菌肺炎患者临床分离菌株中的分布和临床特征的关系.方法:收集2019年8-9月南京医科大学附属无锡人民医院分离的引起肺部感染的50株肺炎克雷伯菌,PCR法检测毒力基因,统计并分析肺炎克雷伯菌毒力基因阳性率及患者的临床分布特征.结果:50株菌株mrkD、ureA基因阳性率为100％,fimH、entB、ybtA基因阳性率分别为86％、96％、64％,未检测出wabG、uge、clbA基因.携带fimH基因肺炎克雷伯菌菌株的感染者较非携带该基因细菌的感染者更易出现咳嗽咳痰、胸闷气短的症状.最常见的组合为fiimH+mrkD+ureA+entB+ybtA,同时检出5种毒力基因的肺炎克雷伯菌感染者临床表现明显,但病死率低.结论:随着毒力基因种类的增加,咳嗽咳痰、胸闷气短症状的比例基本呈上升趋势,发热呈下降趋势.

Multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) are prevalent throughout the world resulting in a major public health burden. In this research, we isolated and identified 28 MDR UPEC from one university hospital in China, investigated MDR and pathogenic mechanisms by PCR, including 55 antibiotic resistance determinants (ARDs) genes, 13 genetic markers of mobile genetic elements (MGEs) and 6 virulence factors (VFs) genes. In these isolates, we identified 23 ARDs genes and 6 genetic markers of MGEs that played a key role in MDR phenotypes. In addition, we found 2 VFs genes, hof Q and omp T, which could be associated with pathogenicity and invasiveness of these strains in urinary tract infections (UTIs).

目的:调查20株耐药粪肠球菌株可能存在的耐药基因状况和菌株间的亲缘关系.方法:收集2015年1月-2018年12月湖北省江汉油田总医院住院患者分离的20株耐药粪肠球菌株,用PCR法检测1种青霉素耐药基因、4种氨基糖苷类耐药基因、6种大环内酯类耐药基因、1种链阳菌素耐药基因、2种四环素耐药基因、3种万古霉素耐药基因、2种季胺类消毒剂耐药基因、1种可移动遗传元件标志基因,最后对检测结果作样本聚类分析(UPGMA法).结果:20株耐药粪肠球菌分别检出1～7种耐药元件基因,包括1种青霉素耐药基因、3种氨基糖苷类耐药基因、2种大环内酯类耐药基因、1种四环素耐药基因、2种万古霉素耐药基因、1种可移动遗传元件标志基因.菌株亲缘关系分析可见20株耐药粪肠球菌可分为A、B2群.B群又可分为B-1、B-2亚群.B-1亚群有2个克隆播散(4号株-14号株-17号株、8号株与11号株),B-2亚群有1个克隆播散(9号株与12号株).结论:青霉素耐药基因、氨基糖苷类耐药基因、大环内酯类耐药基因、四环素耐药基因、万古霉素耐药基因和可移动遗传元件是导致本组粪肠球菌耐药的重要原因.

肺炎克雷伯菌是一种革兰阴性菌,能够引起肺部感染、泌尿道感染及肝脓肿等疾病.作为院内感染的常见细菌,重症监护室患者由于暴露于侵入性设备,引起相关感染的比例会迅速上升[1].肺炎克雷伯菌可分为经典型(cKP)和高毒力型(hvKP)两种.cKP作为一种典型的机会性致病菌,易在免疫力低下、肿瘤及糖尿病患者中引起感染.20世纪90年代台湾地区发现,hvKP能够在普通健康年轻人群中引起肝脓肿,并转移感染引起脑膜炎、眼内炎等疾病.hvKP的高毒力很可能是由它荚膜表达增加引起高黏滞表型引起的,但是高黏滞性的肺炎克雷伯菌并不全都引起严重感染.因此,若要判断菌株为hvKP,需拉丝试验阳性,且临床存在侵袭性感染.目前已发现,菌毛、荚膜、脂多糖(LPS)、嗜铁素及孔蛋白等毒力因子在肺炎克雷伯菌的致病过程中起到了重要作用.本文将对这些已被发现的毒力因子进行论述.

目的 调查一组临床检出的肺炎克雷伯菌可能存在的耐药基因状况,以及各菌株间可能存在的亲缘关系.方法 25株肺炎克雷伯菌均分离自2016-2017年江汉油田总医院医院住院病人标本.菌种鉴定为gyrA基因测序上网BLASTn比对法,用PCR方法检测35种β-内酰胺酶基因,15种氨基糖苷类药物耐药基因,1种喹诺酮类药物耐药相关基因和11种可移动遗传元件.再用DNA测序法分析基因突变.最后对62种耐药元件基因的检测结果作样本聚类分析(UPGMA法).结果 25株耐药肺炎克雷伯菌对碳青霉烯类耐药率低(亚胺培南、美罗培南均只有8％),对其它8种抗菌药物的耐药率较高(40％～96％).每株均检出β-内酰胺类药物耐药相关基因(总阳性率100.00％);有24株菌检出氨基糖苷类药物耐药相关基因(总阳性率96.00％);有20株菌检出喹诺酮类药物耐药相关基因gyrA突变(即突变率为80.00％);每株均检出可移动遗传元件遗传标记基因(总阳性率100.00％).共检出5种β-内酰胺酶基因(bla TEM、bla LAP、bla KPC、bla DHA群、bla OXA-1群),6种氨基糖苷类药物耐药基因(aac(3)-Ⅱ、aac(6')-Ⅰb、ant (3”)-Ⅰ、aph3'-Ⅰ、aadA5、rmtB),20株存在喹诺酮类药物耐药相关基因gyrA第83位密码子发生了同样的突变,且均无第87位密码子的突变.检出8种可移动遗传元件(intⅠ 1、tnpU、tnp513、ISEcp1、IS26、IS903、ISKpn6、trbC),且检出率较高.样本聚类分析显示本组菌可分A与B2个簇群,2个簇群中均有克隆传播.A簇群中有2个克隆,分别为2-3-5-16等4株、13-14等2株;B簇群中有1个克隆,为19-20号2株.结论 多种β-内酰胺类药物耐药基因、氨基糖苷类药物耐药基因,gyrA第83位密码子突变和可移动遗传元件共同导致本组肺炎克雷伯菌对10种抗菌药物的耐药.本组肺炎克雷伯菌3个克隆传播提示可能有医院感染的存在.

大肠埃希菌是医院内常见的条件致病菌,容易造成部分免疫力低下患者形成腹腔感染、尿道炎及血流感染等.近年来,随着抗菌药物的使用日益增多,大肠埃希菌的耐药性也在逐年上升.2014年CHINET耐药性监测网检测结果表明:2005-2014年间临床分离大肠埃希菌的数量增长十分明显,且其对很多常见抗菌药物的耐药性也在逐年升高.其中2014年显示:大肠埃希菌对氨苄西林的耐药率最高,达86％;哌拉西林其次,为71.6％;对头孢唑林、头孢呋辛的耐药率均高于60％;而对临床上一些高档抗菌药物如亚胺培南、美罗培南等,耐药率较低,低于8％[1].因此,研究大肠埃希菌的耐药机制从而指导临床上抗菌药物的合理使用就显得十分关键,现本文将近年来大肠埃希菌对于β-内酰胺类、氨基糖苷类及喹诺酮类药物的耐药机制的研究进展综述如下.

目的 研究获得性肺炎老年患者铜绿假单胞菌分离株的毒力基因和耐药基因的携带状况。 方法 收集2016年1月至2017年12月在山东省日照市中心医院收治的60岁以上获得性肺炎患者痰液中分离的36株铜绿假单胞菌，采用Vitek法对9种抗菌药物进行敏感性试验，采用聚合酶链反应及序列分析法分析9种毒力基因及23种耐药基因(17种β-内酰胺酶类和6种氨基糖苷类耐药基因)，阳性基因测序后直接作BLAST比对。 结果 所有菌株对碳青霉烯类药物均敏感，对三代头孢菌素类、氨基糖苷类和喹诺酮类药物耐药率分别为27.78%、47.22%和30.56%；每株均检出5～8种毒力基因，其中plcH、aprA、algD、exoS、exoT、exoY和toxA检出率极高，高达97.22%～100.00%；检出6种耐药基因，分别为blaCARB、blaDHA、aac(6′)-Ⅰb、aac(6′)-Ⅱ、ant(3″)-Ⅰ和ant(2″)-Ⅰ，分离率为8.33%～27.78%；部分基因的PCR产物测序图已比对确认。 结论 铜绿假单胞菌毒力基因的高携带率与老年人的获得性肺炎关系密切；铜绿假单胞菌未携带blaIMP、blaVIM和blaNDM碳青霉烯类β-内酰胺酶基因与对碳青霉烯类药物敏感密切相关；毒力基因和耐药基因的联合检测能更好的分析老年患者感染病原菌的致病机理。

Objective: To investigate global expression of uropathogenic Escherichia coli in response to the traditional Chinese medicine: Taraxacum mongolicum. Methods: Ten times of MIC of Taraxacum mongolicum decoction was used to culture Escherichia coli strain NB8, total RNA was extracted with rRNA removed using RibioMinus Bacteria Kit, and transcriptome sequencing was performed using Hiseq2000 platform, then gene expression and differential gene expression were analyzed. KEGG enrichment analysis was assigned for all protein sequences in FASTA format by using KAAS. Results: In total, 3,962 differentially expressed genes were found between the treatment group and the control group, including 2,442 up-regulated genes and 500 down-regulated genes. Those up-regulated genes encoded proteins involved in bacterial chemotaxis and flagellar assembly pathway, ABC transporter pathway, Type II secretion system pathway in stress response to Taraxacum mongolicum. Meanwhile, the strong suppression on ribosome pathway, fatty acid biosynthesis, glycolysis and TCA were also observed. Conclusion: Taraxacum mongolicum has significant antibacterial activity on uropathogenic Escherichia coli NB8. Our findings on the transcriptomic level reveal that the response of uropathogenic Escherichia coli to Taraxacum mongolicum decoction is a complex process that involves stress response, DNA damage and repair, metabolic pathway, protein translation and secretion.

患者女,89岁,因膀胱肿瘤、右肾积水于2012年1月30日收住于宁波市第一医院泌尿外科,尿培养示迟钝爱德华菌>100 000 cfu/mL,予氨曲南2.0 g每天两次治疗.2012年2月5日复查尿培养,无细菌、真菌生长,提示尿路感染治愈.用VITEK-2细菌鉴定仪及配套革兰阴性菌鉴定卡鉴定菌种,标准菌株为产酸克雷伯菌ATCC700324. 挑纯培养菌落置入0.5 mL离心管内(内预置200 μg/L蛋白酶K溶液200 μL,56 ℃水浴2 h,改95 ℃水浴10 min,15 000 r/min(离心半径8 cm)离心30 s.上清液即为基因检测的模板液,-20 ℃冰箱保存备用.分子鉴定采用PCR法检测管家基因gyrA.靶基因引物识别序列P1: 5′-CAGGTAGTAGTGACCGTCAC-3′;P2: 5′-AAGCGTGTCGAAGGCATCAG-5′;目的产物长度501 bp.靶基因PCR扩增体系均为每反应体系P1引物1 μL (1.0 μmol/L)、P2引物1 μL(1.0 μmol/L),核苷三磷酸(dNTP) 2 μL(2 mmol/L),10倍缓冲液2 μL[KC1 10 mmol/L,(NH4)2SO4 8 mmol/L,MgC12 2 mmol/L,Tris-HCl(pH值为9.0)10 mmol/L,乙基-苯基-聚乙二醇(NP)40 0.5%,牛血清蛋白(BSA)0.02%],Taq DNA聚合酶1 U(不计体积),超纯水9 μL,模板液5 μL,总反应体积20 μL.PCR扩增热循环参数为:93 ℃预变性2 min;然后93 ℃ 30 s,55 ℃ 30 s,72 ℃ 60 s,循环35周期;72 ℃延长5 min.产物经2%琼脂糖凝胶电泳,出现与阳性对照分子相当的目的条带为阳性.

肺炎克雷伯菌是一种机会性致病菌,可引起社区获得性感染和医院获得性感染. 我们从宁波市第一医院1例36岁男性患者的血液中分离到1株泛耐药肺炎克雷伯菌,进行全基因组测序,并分析其耐药和毒力相关基因.

目的 了解临床分离的携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌中可移动遗传元件(mobile genetic elements,MGEs)存在情况.方法 在2008年11月至2009年7月从住院患者中分离19株携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌,通过分析3种基因gyrA、parC和mdfA序列对菌株进行分子鉴定.采用PCR方法检测4种可移动遗传元件遗传标记基因,分别为:泛宿主性接合性质粒遗传标记trbC、转座子Tn1548遗传标记tnpU、Ⅰ类整合子遗传标记qacE△1-sul1和插入序列共同区遗传标记ISCR1基因;对扩增阳性的4种遗传标记基因PCR产物采用双向测序,测序结果用Chromas软件直接作BLASTSearch比对分析.结果 本组19株gyrA、parC和mdfA基因PCR扩增均阳性,其基因序列经GenBank中的BLASTn程序进行同源性比较分析,证实19株均为肺炎克雷伯菌.19株中,4种可移动遗传元件遗传标记trbC、tnpU、qacE△1-sul1和ISCR1基因阳性率均为100.0％,经对该4种遗传标记基因PCR阳性产物进行测序比对,发现与GenBank中己发布的4种相对应基因trbC、tnpU、qacE△1-sul1和ISCR1序列完全相同(同源性均为100.0％),均得到确认.结论 可移动遗传元件在携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌中广泛存在.

OBJECTIVE To investigate the drug resistance elements of 60 clinical continuous isolates of Escherichia coli and analyze the phylogenetic relationship among the strains.METHODS A total of 60 clinical continuous iso-lates of E.coli were isolated from the patients who were hospitalized from Oct 2015 to Dec 2015,the drug suscep-tibility rates to 12 antibiotics were determined by using K-B method,the related genes resistant to 15 β-lactams,6 aminoglycosides and 3 sulfonamides as well as 4 mobile genetic elements and genetic markers were analyzed by means of polymerase-chain-reaction(PCR)and sequencing analysis;the sample cluster analysis was performed for the results of detection.RESULTS The drug resistance rates of the 60 strains of E.coli to ampicillin,gentamicin, tobramycin,ciprofloxacin and sulfamethoxazole-trimethoprim were more than 50%,the drug resistance rates to other 7 antibiotics were more than 50%,and all of the strains were susceptible to carbapenems.Totally 15 types of drug resistance elements genes were detected in the 60 strains of E.coli,the β-lactams resistance genes were de-tected in 43 strains,with the detection rate 71.7%;the aminoglycosides resistance genes were detected in 33 strains,with the detection rate 55.0%;the sulfonamides resistance genes were detected in 40 strains,with the de-tection rate 66.7%;the mobile genetic elements and genetic markers genes were detected in 42 strains,with the detection rate 70.0%.Totally 6 types of β-lactams resistance genes,4 types of aminoglycosides resistance genes, 3 types of sulfonamides resistance genes and 2 types of mobile genetic elements and genetic marker genes were de-tected.The sample cluster analysis showed that there were 8 suspected clone strains in this group of strains,the strains of the same clone carried with the same drug resistance elements,suggesting that nosocomial infection sex-isted.CONCLUSION blaT EM,aac(3)-Ⅱ,sul1,d f rA17 and intⅠ1 are the leading causes of the resistance of this groups of strains to β-lactams,aminoglycosides and sulfonamides,and the accordance rates of the drug resist-ance phenotypes and genotypes are high.

OBJECTIVE To understand the acquired drug resistance genes in 20 strains of Acinetobacter baumannii and analyze the phylogenetic relationship among the strains .METHODS A total of 20 strains of A .baumannii were isolated from Jan 2015 to Dec 2015 .Totally 31 kinds of acquired resistance genes to beta‐lactams ,12 kinds of ac‐quired resistance genes to aminoglycosides ,and 8 kinds of mobile genetic elements were analyzed by means of pol‐ymerase‐chain‐reaction (PCR) and sequencing analysis ,and the sample cluster analysis (UPGMA) was performed for the detection result .RESULTS This groups of strains were resistant to all of the 11 kinds of antibiotics .Totally 4 kinds of acquired resistance genes to beta‐lactams ,5 kinds of acquired resistance genes to aminoglycosides ,and 5 kinds of mobile genetic elements were detected positive in the strains ,and the positive rates were very high .In ad‐dition ,the acquired resistance genes to beta‐lactams and aminoglycosides ,and mobile genetic elements could be de‐tected positive in each of the strains .The detected resistance elements were divided into 6 positive modes .The sam‐ple cluster analysis indicated that the 20 strains of A .baumannii presented with obvious aggregation ,and strain No .2‐5‐7‐8‐9‐10‐13‐15‐17‐20 (10 strains) ,strain No .11‐12‐16‐19 (4 strains) ,and strain No .1‐4‐6(3 strains) were clonal propagation .CONCLUSION The phenotypes and genotypes of acquired resistance genes to beta‐lactams and aminoglycosides are homologous in this group of A .baumannii ,the mobile genetic elements are the causes of rapid propagation of acquired resistance genes ,and the detection of specified resistance genes can be used as a practical method to study the bacterial resistance .

OBJECTIVE K lebsiella pneumoniae is a common species of pathogen causing nosocomial infections ,the morbidity and drug resistance rate are increased year by year .The mobile genetic elements (MGE) that may carry resistance genes can transfer and spread among the same species or different species ,which may accelerate the drug resistance of the K .pneumoniae to the clinical antibiotics as well as the spread of drug‐resistant strains and bring great difficulties to the treatment .This paper briefly describes the progress of the related researches on MGE carrying with drug resistance genes in the K .Pneumoniae and explores the drug resistance mechanisms of the K .Pneumoniae to the three categories of clinical antibiotics ,includingβ‐lactams aminoglycosides and quinolones .

目的 检测1株多耐药大肠埃希菌NB8株的遗传学背景.方法 病原分离自2012年4月宁波市第一医院住院患者尿液,作16S rDNA和gyrA基因测序、BLASTn比对确认为大肠埃希菌,采用Illumina HiSeq与Ion Torrent PGM两种大规模并行测序仪进行全基因组分析,再行人工测序.最后NB8株和9株已完成全基因组测序的大肠埃希菌耐药基因的比较基因组学研究.结果 多耐药大肠埃希菌NB8株全基因组测序得到一条推定的染色体序列,长4 550 369 bp(内含14个缺口),得到一条推定的质粒序列,长635 377 bp(内含33个缺口).从NB8株全基因组数据中挖掘出了β-内酰胺类、氨基糖苷类、喹诺酮类、大环内酯类、磺胺类、四环素类、多粘菌素的耐药基因,并挖掘出接合性质粒、转座子、插入序列、整合子的遗传标记,以及5大类外排泵基因.结论 多耐药大肠埃希菌NB8株遗传学背景明确,主动外排机制增强也会导致或者增强NB8株的耐药性.质粒、转座子和整合子等可移动遗传元件可以使细菌的耐药性得以快速传播,使受体菌表现为多重耐药.

We present the draft genome sequence of a Klebsiella pneumoniae carbapenemase-producing sequence type 2357 (ST2357) strain, NB60, which contains drug-resistant genes encoding resistance to beta-lactams, fluoroquinolones, aminoglycosides, trimethoprim-sulfamethoxazole, colistin, macrolides, and tetracycline. Strain NB60 was isolated from human blood, making it an important tool for studying K. pneumoniae pathogenesis.

OBJECTIVE To investigate the distribution of virulence genes (pspA ,pspC ,lytA ,lytB ,rrgA ,nanA , pav ,iga ,ply and hysA ) among Streptococcus pneumoniae isolated from clinical patients so as to provide impor‐tant reference for the research on the roll of virulence genes in the pathogenesis of the S .pneumoniae and the de‐velopment of new vaccine .METHODS Totally 20 strains of S . pneumoniae were isolated from patients in Wuxi People′s Hospital from Jan .2013 to Dec .2014 .Polymerase chain reaction(PCR) was used to screen for the ten target virulence genes .The positive rate of the virulence genes and the relationship with the bacterial drug resist‐ance were statistically analyzed .SPSS 17 .0 was explored to make statistical analysis .RESULTS The positive rates of ten virulence genes in 20 clinical isolates were respectively 0 ,0 ,45 .0% ,60 .0% ,35 .0% ,60 .0% ,75 .0% , 80 .0% ,55 .0% ,and 55 .0% .Among them ,positive rates of pav and iga were relatively higher .The positive rates of pav (64 .5% ) and rrgA (100 .0% ) in erythromycin resistant strains were significantly higher than those of non resistant strains (P<0 .05) .CONCLUSION The ten virulence genes each have different levels of expression in S .pneumoniae in this region .The detection rate of pav and iga was relatively higher ,which may become new vaccine candidate molecules .RrgA and pav may be related to erythromycin resistance .

OBJECTIVE To investigate all kinds of exotoxin genes carrying status in Staphylococcus aureus isolates in burn patients ,so as to provide the basis for the treatment for S .aureus infection .METHODS S .aureus were i‐solated from the samples of our hospital burn patients from Jan .to Jun .2015 ,using Vitek‐2compact automatic i‐dentification device to give strain identification ,using PCR to analysis exotoxin genes set1‐26 and set30‐40 ,and u‐sing Q type to have cluster analysis .RESULTS After using 37 kinds of exotoxin genes to detect 20 strains of S . aureus ,only 1 strain was not detected any gene ,and the remaining 19 strains had positive findings .Exotoxin genes only set2 ,set 4 ,set16 ,set23 ,set37 ,set39 ,etc had non positive findings ,the positive rates for the rest were within 5 .0% ~ 95 .0% .There were 24 genes with positive rates more than 50% ,15 genes more than 75% . The detection results of exotoxin genes had diversity with 17 patterns .Cluster analysis showed that the 20 strains of S .aureus could be classified into big and small families .CONCLUSION There are 20 strains of S .aureus exo‐toxin genes with high positive rates ,which may be related to the impetigo in burn wound and cellulitis .

Escherichia coli NB8 is a clinical pyelonephritis isolate. Here, we report the draft genome sequence of uropathogenic E. coli NB8, which contains drug resistance genes encoding resistance to beta-lactams, aminoglycosides, quinolones, macrolides, colistin, sulfonamide-trimethoprim, and tetracycline. NB8 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis.

OBJECTIVE To investigate the active screening of adhesion toxin and superantigen genes in Staphylo‐coccus aureus isolated from ICU patients so as to provide guidance for development of prevention and control pro‐grams .METHODS From 2013 to 2014 ,a total of 46 strains of S .aureus that were isolated from nasal swab and given active screening were collected ,then the strains were identified by using chromID S .aureus plate sreening and Vitek‐2 bacterial identification system of BioMérieux ,France ,and 16 kinds of adhesion toxin genes and 20 kinds of superantigen genes were detected with the use of polymerase‐chain‐reaction (PCR) .RESULTS Totally 9 kinds of adhesion toxin genes and 10 kinds of superantigen genes were detected in the 46 strains of S .aureus;the total detection rate of the adhesion toxin genes was 100 .0% ,and total detection rate of the superantigen genes was 65 .0% .CONCLUSION The high detection rate of the adhesion toxin genes and superantigen genes in the S .aureus is the leading cause of pulmonary or bloodstream infection in the ICU patients .