医院感染阴沟肠杆菌的耐药性及基因型分析

了解中南大学湘雅三医院重症监护病房(ICU)医院感染阴沟肠杆菌的耐药谱及分子流行病学特征,指导临床合理用药。基因分型采用优化反应体系的随机扩增多态性DNA(RAPD)法,耐药分析选用K-B法。RAPD分型将28株阴沟肠杆菌分为11种株型,耐药谱则将其分为12种不同的耐药型。基因分型和耐药分析显示ICU有多重耐药的阴沟肠杆菌株爆发流行现象,它为控制阴沟肠杆菌的医院内感染、追踪传染原、切断传播途径提供遗传学信息,指导临床医生选用敏感有效的抗生素。     

随机扩增多态性DNA分析院内感染的铜绿假单胞菌

目的 运用随机扩增多态性DNA(RAPD)基因分型技术监测铜绿假单胞菌(PA)的医院感染情况,了解耐药表型与RAPD基因型关系,为确定院内交叉感染发生及院感控制提供依据.方法 对10例ICU患者连续多次采样分离出的48株PA进行RAPD基因分型和药物敏感性试验.结果 48株PA共分出9个基因型.6例患者为单一RAPD型PA菌株感染,4例患者检出2种RAPD 型,有3组患者分别检出同型RAPD.耐药性最高的是环丙沙星(75%),其次是左氧氟沙星(73%),耐药性最低的为美洛培南(31%).结论 耐药表型与RAPD基因型之间不存在相关性;RAPD分型快速简单,对于流行病学调查确定院内交叉感染或流行具有重要意义.     

全耐药鲍曼不动杆菌分子分型和分子流行病学调查

重复片段引物PCR和随机扩增多态性DNA(RAPD)技术对临床分离全耐药不动杆菌分子分型,并进行流行病学调查.从ICU病房感染多重耐药不动杆菌患者的标本分离不动杆菌,碱裂解法提取全基因组,重复片段引物PCR(Rep-PCR)和随机引物扩增(RAPD),对8株临床分离的全耐药菌基因分型,并与生物学分型和质粒分型比较,调查医院流行全耐药菌的基因型.结果显示,8株分离菌经两对重复片段引物分型可分为6种和4种基因型,经随机引物分型为4种和3种基因型,经质粒分型可分为2种基因型,生物学分型归属为1种表型.PCR方法用于全耐药不动杆菌分子分型简便易行,重复性好,适合医院感染流行病学调查,本医院同一部门出现多种基因型,各科室间不存在交叉传染.     

鲍曼不动杆菌随机扩增多态性DNA法基因分型的研究

目的 利用随机扩增多态性DNA(RAPD)技术对鲍曼不动杆菌进行基因分型建立DNA指纹图谱,调查该菌在各临床科室的流行情况,并将其与药敏谱进行比较.方法 随机收集中南大学湘雅二医院2007年9月到2008年9月分离出的86株鲍曼不动杆菌,采用WHO推荐的K-B法对鲍曼不动杆菌进行药物敏感试验,建立药敏谱,同时利用随机扩增多态性DNA法(RAPD)技术进行基因分型,建立DNA指纹图谱,对二者进行比较,然后结合药敏谱和指纹图谱分析各临床科室鲍曼不动杆菌的感染情况.结果 药物敏感试验将86株鲍曼不动杆菌分为47型,RAPD技术将其分为14型,其中A、F、D、B和L型为5种优势型,其菌株数分别为22、15、11、9和7株.本院ICU,老年病科,神经内科,呼吸内科,神经外科鲍曼不动杆菌检出率较高.结论 RAPD分型方法优于药敏分型,其在流行病学研究上更能证实菌株的相关性,在早期发现和预防感染暴发流行中起重要作用.     

采用RAPD分析臭鼻克雷伯菌临床菌株的基因型

臭鼻克雷伯菌是重要的条件致病菌 ,常引起老人及婴幼儿重症护理病房的交叉感染及爆发流行 ,对其进行流行病学监测有重要的医学意义。用随机引物聚合酶链式反应 (RAPD)对 37株臭鼻克雷伯菌临床菌株基因组DNA进行分型 ,分析扩增产物的指纹图谱。实验结果表明 ,37株臭鼻克雷伯菌RAPD后可出现不同的指纹图谱 ,根据特异泳带C ,J ,F ,Q的有无 ,可将其分 7型 (I～VII)。RAPD可用于检测臭鼻克雷伯菌基因组DNA的异质性 ,是医院内感染分子流行病学研究的好方法。     

鲍曼不动杆菌临床分离株的耐药性及同源性分析

目的 通过对重庆医科大学附属第二医院近4年临床分离的103株鲍曼不动杆菌的耐药性和同源性分析,了解该院鲍曼不动杆菌的耐药性特点及院内感染流行状况.方法 2008年至2011年间该院临床科室分离的103株鲍曼不动杆菌,进行药敏试验并对耐药性分析;提取细菌基因组DNA,以随机扩增DNA多态性(RAPD)方法进行基因分型.结果 药敏试验结果显示分离出的103株菌呈现出高耐药率及多重耐药率的特点,其中对左氧氟沙星和亚胺培南的耐药性最低,分别为69.9％和57.3％.103株多重耐药鲍曼不动杆菌用RAPD分型共分为16种基因型:A～P,其中A型84株,为主要流行型别;B型3株;C型、G型各2株;其余12型各1株.结论 该院鲍曼不动杆菌具有多重耐药及高耐药率,可能存在以A型鲍曼不动杆菌克隆株传播方式的院内流行,临床上应加强对A型鲍曼不动杆菌的监控,采取有效的措施以预防院内感染的爆发流行.     

IRS-PCR在鲍曼不动杆菌基因分型研究中的应用

探讨低频限制性位点聚合酶链反应(infrequent-restriction-site polym erase chain site,IRS-PCR)在鲍曼不动杆菌基因分型中的应用。用IRS-PCR分别扩增上海、哈尔滨两地各20株鲍曼不动杆菌DNA稀有限制区旁序列并分型。与RAPD法比较两种基因分型方法的分型率、分辨力、重复性。IRS-PCR可将上海株分13个型别,RAPD分为19个型别,两种方法分型的一致率为60%(12/20);IRS-PCR将哈尔滨株分成19个型别,RAPD分为20个型别,两种方法分型的一致率为90%(18/20)。与RAPD相比,IRS-PCR的条带数多,分辨力强,重复性好。故IRS-PCR可用于鲍曼不动杆菌的分型研究,也是一种有价值的分子流行病学研究工具。     

鲍曼不动杆菌IRS-PCR基因分型研究

目的 用低频限制性位点聚合酶链反应(IRS-PCR)对鲍曼不动杆菌进行基因分型,分析基因型与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学中的作用.方法 随机收集2008年8月至2009年8月临床分离的73株鲍曼不动杆菌,采用K-B法进行药物敏感试验确定鲍曼不动杆菌耐药谱;同时利用IRS-PCR对此73株鲍曼不动杆菌进行基因分型;并分析IRS-PCR分型与鲍曼不动耐药谱的关系;结合IRS-PCR分型结果与73株鲍曼不动杆菌感染病例的临床资料,分析在此时间段鲍曼不动杆菌在我院流行感染的情况.结果 药物敏感试验将73株鲍曼不动杆菌菌株分为A1(19株全耐药型)和A2 ～ A31(54株耐药谱型)31个药敏谱.IRS-PCR法将其分为A～W共23个基因型,其中A、C、B、D和E型为5种优势菌株,分别为14、11、10、8和6株.对比研究发现A1型菌株(15/19)主要集中在基因型A、C、D内,而基因型B包含A15型耐药菌株9株(69.2％),基因型E包含A3型耐药菌株3株(42.9％).A基因型在院内特别是ICU中心引起2次爆发流行,而C和D型主要在呼吸内科引起感染.结论 IRS-PCR基因分型与药敏分型有较高的一致性,且IRS-PCR基因分型在早期发现和预防感染暴发流行方面优于药敏分型.     

重症监护病房金黄色葡萄球菌耐药性与流行病学分析

为了解重症监护病房感染金黄色葡萄球菌的耐药性及流行状况,收集重症监护病房2007年9～12月临床分离的金黄色萄萄球菌42株,纸片扩散法检测其对10种抗生素的耐药率,随机引物扩增PCR(Ran- dom amplified polymorphic DNA,RAPD)检测其流行状况。42株金黄色葡萄球菌对氨苄西林的耐药率最高,没有检测到对万古霉素耐药的金黄色葡萄球菌。35株金黄色葡萄球菌为耐甲氧西林的金黄色葡萄球菌(MR- SA),7株为甲氧西林敏感的葡萄球菌(MSSA),除万古霉素外,MRSA对其他9种抗生素的耐药率比MSSA高,42株金黄色葡萄球菌经RAPD分型分为5个基因型,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ型分别占31.0%、38.1%、14.3%、9.5%、7.1%。重症监护病房临床金黄色葡萄球菌分离株对多种抗生素具有高耐药性,其感染基因型以Ⅰ、Ⅱ型为主。     

广州市公共场所中央空调冷却塔水中军团菌mip基因分型

【目的】研究广州市公共场所中央空调冷却塔水中军团菌的基因特征和优势型别。【方法】采用军团菌巨噬细胞感染力增强因子(Macrophage infectivity potentiator,mip)基因分型方法。提取广州市2008-2010年分离的140株(119株嗜肺,21株非嗜肺)军团菌基因组DNA,针对mip基因进行PCR扩增并测序,将核苷酸序列上传至欧洲军团菌感染工作组(EWGLI)数据库进行比对,得到mip型别,并构建系统发育进化树。【结果】140株军团菌均可扩增出700 bp左右的目的条带。119株嗜肺军团菌分为10个mip型别,L.pneumophila-phil-1为优势型别,占52.9%(63/119);21株非嗜肺军团菌分为6个mip型别,L.feeleii-D3131为优势型别,占47.6%(10/21)。【结论】广州市公共场所中央空调冷却塔水中军团菌具有多样性,mip分型技术可用于军团菌的快速基因分型。     

Serologic and genetic analysis of group B streptococci isolated from adult patients

Serologic and genetic typing with RAPD (Random Amplified Polymorphic DNA) method was used for epidemiologic analysis of GBS. 125 strains isolated from various clinical samples from adult patients were tested. In serologic typing seven serotypes have been found. Serotypes III and R were the most often encountered, containing 37,6% and 20,8% of samples. There was no dependence between serologic type and disease process. Optimalisation of RAPD reaction parameters was based on the standard strains of GBS. In the group of strains tested with the use of RAPD method, eleven genetic profiles were found, with prevalence of profile B (25,8%). Five other profiles occured with similar frequency (8,8% - 12,8%). Among streptococci isolated from patients with the infection of genitourinary tract, great differentiation in the genetic profiles of the strains has been found. Each serologic type contained various genetic profiles. Genetic variety showed by RAPD method indicates the raised ability of this technique to find differences among isolates of GBS.     

Utility of a pre-optimized kit for random amplified polymorphic DNA in typing Candida albicans

The utility of a pre-optimized kit for random amplified polymorphic DNA (RAPD) was assessed in typing diverse strains of Candida albicans from epidemiologically unrelated inpatients (interpatient analysis) and in detecting clonal variations that maybe present within individual patient isolates (intrapatient analysis). Stool samples from inpatients were cultured on Inhibitory Mold agar. Nine individual colonies from all patients with > or =9 colonies of C. albicans (n = 18) were selected, frozen, and karyotyped using CHEF genomic DNA plug kits and CHEF-DRIII. Each of the selected colonies was then analyzed by RAPD, utilizing the selected kit, with 6 primers. Interpatient analysis revealed 9 karyotypes and 17 RAPD composites. RAPD discrimination was significantly better (p < 0.001). Intrapatient analysis revealed 34 (21%) and 33 (20.4%) variants among 162 colonies tested by RAPD and karyotyping, respectively. The results were discordant in 25 variants, all with differences of 1-3 bands. These results illustrate that this pre-optimized kit for RAPD provides excellent discrimination of genetically unrelated strains. Its performance in delineating subtle clonal differences was comparable with karyotyping; both methods failed to detect all minor genetic variations. The ease of use and quick turnaround time of this kit offer a practical and reliable method for typing diverse strains of C. albicans, but may be inadequate for assessing microevolution.     

Identification of Glomerella cingulata f. sp phaseoli recombinants by RAPD markers

We examined the capacity of strains of Glomerella cingulata f. sp phaseoli fungus (Colletotrichum lindemuthianum sexual stage) to form recombinants, using random amplified polymorphic DNA (RAPD). Crosses of all possible combinations between strains 40, 42, 20, 21, 22, 23, 24, 25, and 26 were made on Petri dishes using M3 culture medium. The 42 x 21 cross produced the largest number of perithecia and five asci; the respective ascospores were isolated. RAPD analysis was performed on the parents and descendants. The 62 polymorphic RAPD bands obtained were used to assess the genetic similarity using the method of Sorence and Dice and clustering analysis in the form of a dendrogram by the UPGMA method. The RAPD markers allowed identification of recombinants from the cross between strains 42 and 21 of G. cingulata f. sp phaseoli and 40 ascospores presented 63 and 49% genetic similarity with parents 2 (strain 42) and 1 (strain 21), respectively.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Potential of Three-Way Randomly Amplified Polymorphic DNA Analysis as a Typing Method for Twelve Salmonella Serotypes

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

香菇菌株分子鉴别技术的分辨率比较

本研究运用酯酶同工酶、RAPD、IGS1和IGS24种方法鉴别2个野生香菇菌株和19个栽培香菇菌株,并比较这4种鉴别方法的分辨率,结果表明:16条酯酶同工酶带中有15条具有多态性,将21个供试菌株分成11个类型,分辨率为0.92;10个随机扩增引物共扩增出86个DNA片段,其中95.3%具有多态性,将21个供试菌株分成16个类型,分辨率为0.97;IGS1将21个供试菌株分成7个类型,分辨率为0.81;IGS2将21个供试菌株分成7个类型,分辨率为0.73。这4种鉴别方法中RAPD的分辨率最高,与这4种鉴别方法的综合分析结果相同,因此RAPD可以作为香菇菌株鉴别的可靠依据。     

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD-PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74.3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region-R sizes. There was a strong association between RAPD type and the clfA region-R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87.5%) coproduced toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma-haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.     

Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.     

Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis

Aims:  To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location.
Methods and Results:  Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi , the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia.
Conclusions:  The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical.
Significance and Impact of the Study:  This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains.