铜绿假单胞菌随机扩增多态性DNA指纹法基因分型研究

建立了铜绿假单胞菌（ＰＡ）随机扩增多态性ＤＮＡ（ＲＡＰＤ）指纹图基因分型方法，并应用于流行病学相关或不相关的７５个ＰＡ菌株的基因分型。分型率为１００％。３２株新生儿暴发感染菌株，可分成４个血清型和５个ＲＡＰＤ谱型，其中２５株属于同一谱型。在８例患者的垂直追踪菌株中，除有１例其第１和第３个分离株的血清型和ＲＡＰＤ谱型均相同，但与第２个分离株的血清型和ＲＡＰＤ谱型不同外，其余７例前后菌株的谱型相同。１３株散发菌株可分成３个血清型和１０个ＲＡＰＤ谱型；１２株流行病学无关菌株的ＲＡＰＤ谱型均不相同。本研究结果表明，ＲＡＰＤ指纹图基因分型法具有分型率高、分辨力强、比较快速简便等优点，并且不需要已知核酸序列，是在分子水平上对微生物感染的病原学、发病机理及流行病学研究的较理想的方法。     

伤寒沙门菌PFGE分子分型及耐药性研究

目的对深圳市2004—2011年伤寒沙门菌进行脉冲场凝胶电泳（PFGE）分子分型及耐药性分析,为临床用药和追踪传染源提供依据。方法采用K-B法对42株伤寒沙门菌进行20种抗菌药物敏感性测试,并用PFGE对其进行分子分型。结果42株伤寒沙门菌株对氨苄西林、阿莫西林／克拉维酸、氨苄西林／舒巴坦、头孢噻吩、头孢他啶、头孢曲松、头孢吡肟、头孢西丁、阿米卡星、庆大霉素、卡那霉素、环丙沙星、左旋氧氟沙星、复方新诺明、甲氧苄啶、氯霉素和四环素17种药物的敏感率均超过90％,对萘啶酸耐药率最高达61．5％。42株伤寒沙门菌株可分为SZ0001～SZ0028共28个PFGE型别,其中流行优势型为SZ0023型别。结论结合药物敏感试验结果和PFGE分子分型结果可以判断伤寒疫情的优势株型。     

食源性沙门氏菌血清分型及分子分型分析

目的:对贵阳市2015年至2020年33株食源性沙门氏菌进行血清分型和PFGE分子分型,评价不同血清型沙门菌的亲缘性,为贵阳市沙门菌感染的防控提供参考数据.方法:对目标沙门氏菌进行血清凝集确定其血清型,并使用脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)进行分子分型.结果:血清分型共分出5种血清型,其中优势血清型为鼠伤寒沙门(46％)与德尔卑沙门(21％).分子分型33株菌株中4株分型失败,剩余29株共获得24种带型,相似性达100％ 的有4组(有一组为不同年份肠炎沙门),鼠伤寒优势带型为P15(25％),德尔卑沙门氏菌优势带型为P20(33.3％).结论:本实验沙门菌PFGE分型总体呈多态性,不同年份肠炎沙门分型一致提示存在污染源清除不彻底问题.     

阴沟肠杆菌基因分型方法的比较研究

细菌分型的方法多、应用灵活、对不同的菌属分辨力不同 ,实验室之间缺乏统一的应用标准 ,造成不同实验室之间结果无法进行比较。阴沟肠杆菌己在院内感染中居于越来越重要的地位 ,有必要对其进行分型的研究。本实验对 2 1株阴沟肠杆菌进行多种基因分型。对比了ＲＡＰＤ、ＲＥＰ ＰＣＲ、ＥＩＲＣ ＰＣＲ和ＰＦＧＥ目前常用的几种细菌基因分型方法对阴沟肠杆菌分型的分辨力 ,对其分辨力、分型率、可操作性作了综合的评价。对细菌最佳分型方法的比较探讨 ,可以为实验室之间结果解释标准化提供思路。材料和方法实验菌株 :阴沟肠杆菌 ,16株来自解…     

杭州地区伤寒及甲型副伤寒沙门菌流行菌株分子特征的研究

目的 了解并确定2002-2008年杭州地区伤寒沙门菌、甲型副伤寒沙门菌优势菌株的分子特征.方法 采用脉冲场凝胶电泳(PFGE)、多位点串联重复序列分析(MLVA)或多位点序列分型(MLST)对2002-2008年杭州地区分离的31株伤寒沙门菌、404株甲型副伤寒沙门菌进行分型并分析.结果 404株甲型副伤寒沙门菌可分为6个PFGE型,P1型和P2型属于同一个克隆系,99.0％ (400/404)菌株属于该克隆系,其中P1型菌株占该克隆系菌株的93.3％ (373/400).31株伤寒沙门菌株存在高度多样性,可分为14个PFGE型、28个MLVA型(分辨率90.3％)、3个MIST型.根据MLVA各型靶位点多态性差异,本地区流行的伤寒沙门菌株与东南亚地区菌株相近,但与欧洲菌株差距较大,呈高度多态性的可变串联重复序列(VNTR)位点TR1、TR2、Sal02可作为本地区伤寒沙门菌株的检测标志.伤寒沙门菌株MLST型别包括了目前国际上发现的所有3个型别,但以ST2型为主(23/31,74.2％).结论 近年杭州地区甲型副伤寒疫情由同一亚系菌株感染所致,但本地区流行的伤寒沙门菌株呈现高度多样性.     

流感嗜血杆菌表型和脉冲场凝胶电泳分型研究

目的 应用生物学分型、血清学分型和脉冲场凝胶电泳技术(PFGE)分析临床分离流感嗜血杆菌的表型和分子分型特征,了解流感嗜血杆菌的分子流行病学规律.方法 1988年和2004-2007年期间,成都市儿童医院从患者的下呼吸道分泌物分离培养273株流感嗜血杆菌,菌株的分离培养与鉴定参照美国<临床微生物手册>.菌株的生物学分型和血清学分型分别参照Kilian和Pittman分类法.应用PCR方法对所有的菌株进行荚膜鉴定和血清学分型鉴定.随机选择100株流感嗜血杆菌菌株,应用PFGE技术进行分子分型研究.结果 273株流感嗜血杆菌可分为8个生物型.17.6%(48/273)的菌株为生物型I型,43.6%(119/273)为生物型Ⅱ型,22.7%(62/273)为生物型Ⅲ型,7.3%(20/273)为生物型Ⅳ型,5.9%(16/273)为生物型V型,0.4%(1/273)为生物型Ⅵ型,1.8%(5/273)为生物型Ⅶ型,0.7%(2/273)为生物型Ⅷ型.99.6%(272/273)的菌株为无荚膜的、血清学不可分型的流感嗜血杆菌,1株为血清型f型.100株流感嗜血杆菌经PFGE分析,可分为96个PFGE基因型,93株菌株各自代表一个PFGE基因型.基因型的分布与菌株的分离时间无明显的相关性.结论 引起儿童下呼吸道感染的流感嗜血杆菌主要是无荚膜的、不可分型流感嗜血杆菌菌株,生物型主要是Ⅰ、Ⅱ和Ⅲ型.血清学不可分型流感嗜血杆菌菌株PFGE基因型呈现明显的多态性,菌株的分子多态性可能是流感嗜血杆菌感染在成都市没有形成暴发与流行的分子流行病学原因;PFGE基因分型与生物学分型和血清学分型比较,具有很强的菌株分辨能力,是流感嗜血杆菌感染流行病学研究适用的分析方法.     

MLVA和Spoligotyping用于西藏地区216株结核分枝杆菌临床分离株的基因分型研究

目的评价间隔区寡核苷酸分型（Spoligotyping）及多位点可变数量串联重复序列分析（MLVA）两种分型方法在西藏地区结核病分子流行病学中的应用。方法收集西藏地区结核分枝杆菌（Mycobacteriumtuberculosis）临床分离株，应用Spoligotyping及MLVA两种分型方法进行比较分析。结果共在西藏地区收集到216株结核分枝杆菌临床分离株，采用Spoligotyping分型方法，216株菌可分为3个基因群13种基因型，其中最大的1个基因群即北京家族（8eijingfamily）含有195株菌，占90．28％。北京家族菌株中，有BCG接种史者占45．64％（89／195），无BCG接种史者占54．36％（106／1195），两者间的差异无统计学意义（X^2=0．059，P〉0．05）。采用MLVA分型方法，216株菌可分成19个基因群108种基因型，其中80种基因型只有1株菌，占37．03％（80／216），另有136株菌表现出28种基因型，成簇数为28，占62．96％（136／216）。在20个VNTR位点的等位基因多态性发现Miru31位点的多态性最高，多态性指数（h）达到0．77，而Mtub29、Mtubl2位点的多态性较差，都低于0．05。其中Mtuh02位点可鉴别北京家族和非北京家族，它鉴别的北京家族与Spoligotyping鉴别的北京家族符合率达到100％。结论西藏地区结核分枝杆菌具有明显的接引多态性，其主要流行型为北京家族。北京家族菌株与BCG接种无相关性。应用Spoligotyping和MLVA两种分型方法进行结核病流行病学研究，将提高结核病的流行病学调查和病原学监测效果。     

杭州地区临床和食品来源德尔卑沙门菌耐药特征与基因组分析

目的了解杭州地区临床和食品来源德尔卑沙门菌的耐药特征, 并进行溯源分析。方法对2015—2020年杭州地区分离的60株德尔卑沙门菌进行药敏分析、脉冲场凝胶电泳(PFGE)分型和全基因组测序, 并下载公共数据库基因组进行比较;利用测序数据对菌株进行多位点序列分型(MLST)、核心基因组多位点序列分型(cgMLST)和耐药基因扫描, 并构建基于单核苷酸多态性(SNP)位点的系统发育树。结果杭州地区德尔卑沙门菌临床株和食品株对28种药物的耐药率差异均无统计学意义, 多重耐药率为76.7%(46/60);所有菌株均检出氨基糖苷乙酰转移酶基因aac(6′)-Iaa和磷霉素耐药基因fosA7;60株德尔卑沙门菌共分为46种PFGE带型、53种cgMLST(HC2)型别, 除1株为ST3220型外, 其余均为ST40型;基于439株德尔卑沙门菌(包括60株杭州菌株和379株公共数据库菌株)SNP位点构建的系统发育树显示:部分杭州菌株与东南亚菌株进化距离较近, 提示可能存在跨境传播, 食品株主要来自猪肉和水产类;其他杭州菌株与北京、广州、湖北、重庆等省市的菌株距离较近, 提示可能存在跨省传播, 食品株...     

ICU中MRSA耐药基因检测及其PFGE分型

目的对耐甲氧西林金黄色葡萄球菌（MRSA）菌株进行分子分型,探讨ICU中MRSA医院感染的特点和流行规律。方法采用表型筛选和PCR扩增mecA基因方法鉴定MRSA菌株。脉冲场凝胶电泳方法（PFGE）进行分子分型。结果12株金黄色葡萄球菌表型筛选为MRSA,MRSA产生A型、B型、C型和D型4种耐药表型,优势耐药模式是A型（75．0％）,MRSA对苯唑西林、阿莫西林／克拉维酸和氨苄西林／舒巴坦等10种抗生素产生100％耐药性,11株MRSA携带mecA基因,携带率为91．7％,PFGE指纹图谱分两型,分别为R1型和R2型,11株MRSA为R1型（91．7％）,R1型各株间相似度为100％。结论ICU可存在MRSA爆发流行,MRSA产生多重耐药性（MDR）,MRSA携带mecA基因可表现为MDR,PFGE分型是理想的分子流行病学溯源手段。     

广东省腹泻病例非伤寒沙门菌耐药谱和PFGE分型研究

目的 了解广东省腹泻病例非伤寒沙门菌的耐药状况,并对多重耐药菌株进行脉冲场凝胶电泳(PFGE)分型研究.方法 利用血清学方法对2009-2011年广东省腹泻病例分离的非伤寒沙门菌进行分型,并用CLSI( Clinical and Laboratory Standards Institute)推荐的纸片法对分离的沙门菌株进行抗生素敏感性检测,采用PFGE进行分型研究.结果 91.76％ (256/279)的鼠伤寒沙门菌对3种及以上抗生素耐药,40株鼠伤寒沙门菌同时对9种及以上抗生素耐药,其中有3株对全部12种抗生素耐药.96.91％ (94/97)的I4,5,12:i:-沙门菌对3种及以上抗生素耐药,9株14,5,12:i:-沙门菌同时对9种及以上抗生素耐药,其中有1株对全部12种抗生素耐药.47％(47/100)的肠炎沙门菌对3种及以上抗生素耐药,其中1株对9种及以土抗生素耐药.环内沙星的耐药率为4.27％( 27/632),其中,17株为鼠伤寒沙门菌,6株为14,5,12:i:-沙门菌.31.96％( 202/632)的沙门菌对环丙沙星显示为中介敏感性.这些多重耐药的菌株和具有相同耐药谱的菌株PFGE指纹图谱不完全一致,PFGE型别存在明显的遗传多样性.结论 广东省非伤寒沙门菌多重耐药现象严重.多重耐药菌株的PFGE型别多样,存在明显的遗传多样性.继续加强耐药监测和控制抗生素的合理使用刻不容缓.     

Comparative Analysis of Infrequent-Restriction-Site PCR and Pulsed-Field Gel Electrophoresis for Epidemiological Typing of Legionella pneumophila Serogroup 1 Strains

Two methods were compared for the analysis of 48 unrelated and epidemiologically related Legionella pneumophila serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for XbaI and PstI restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with SfiI. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of L. pneumophila from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, ≥0.996) tool for typing of Legionella. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.     

Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile

Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficile infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods-AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)-to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile.     

Evaluation of Typing of Vibrio parahaemolyticus by Three PCR Methods Using Specific Primers

Vibrio parahaemolyticus is a halophilic bacterium frequently involved in human outbreaks of seafood-associated gastroenteritis. For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen; however, these methods are mostly labor-intensive and time-consuming. In this work, we designed and evaluated three rapid PCR typing methods for this pathogen using primers designed on the basis of the following specific sequences: conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC). Typing patterns and clustering analysis indicated that these methods apparently differentiated V. parahaemolyticus strains from reference strains of interspecific Escherichia coli, V. cholerae, and V. vulnificus and were also valuable in subspecies typing of this pathogen. Forty domestic strains of V. parahaemolyticus, representing a wide range of PFGE patterns, were grouped into 15, 27, and 27 patterns, with discrimination indexes of 0.91, 0.97, and 0.98, by RS-, REP-, and ERIC-PCR, respectively. The discriminative abilities of these PCR methods closely approached or even exceeded those of PFGE and ribotyping. REP-PCR is preferable to ERIC-PCR because of the greater reproducibility of its fingerprints, while RS-PCR may be a practical method because it generates fewer amplification bands and patterns than the alternatives.     

Performance of phenotypic and genotypic methods to determine the clinical relevance of serial blood isolates of staphylococcus epidermidis in patients with septicemia

Five typing methods, including biotyping (API ID32; BioMérieux, Marcy l'Etoile, France), quantitative antibiogram typing based on actual zone sizes, plasmid typing, randomly amplified polymorphic DNA (RAPD) analysis (with primer M13 and primer set ERIC-2-1026), and pulsed-field gel electrophoresis (PFGE), were compared with a previously performed method of DNA fingerprinting by AFLP (amplified fragment length polymorphism analysis) for their performance in the typing of blood isolates of Staphylococcus epidermidis. Sixteen epidemiologically unrelated strains and 11 sets of four blood culture isolates from 11 patients with septicemia were used. The stabilities and reproducibilities of the patterns, the discriminatory capacities of the methods, and the ability to apply the methods to blood culture isolates were used as performance criteria. All strains tested were typeable by each method, and the patterns were stable and reproducible. The numbers of different types within the collection of 16 epidemiologically different isolates were 5 by biotyping, 14 by antibiogram typing, 4 by plasmid typing, 9 by the RAPD assay (combination of results with primer M13 and primer set ERIC-2-1026), and 16 by PFGE. Within the 11 sets of four blood culture isolates the types found by quantitative antibiogram typing, plasmid typing, and PFGE were unique for each set, whereas by biotyping and RAPD analysis some types were observed in more than one set. The results of biotyping did not correspond with the results of the other methods or the results of AFLP. For 6 of the 11 sets, the results of all methods except those of biotyping corresponded completely. Quantitative antibiogram typing, PFGE, and AFLP proved to be the most accurate of the six typing methods tested.     

Molecular Typing of Multiple-Antibiotic-Resistant Salmonella enterica Serovar Typhi from Vietnam: Application to Acute and Relapse Cases of Typhoid Fever

The rate of multiple-antibiotic resistance is increasing among Salmonella enterica serovar Typhi strains in Southeast Asia. Pulsed-field gel electrophoresis (PFGE) and other typing methods were used to analyze drug-resistant and -susceptible organisms isolated from patients with typhoid fever in several districts in southern Vietnam. Multiple PFGE and phage typing patterns were detected, although individual patients were infected with strains of a single type. The PFGE patterns were stable when the S. enterica serovar Typhi strains were passaged many times in vitro on laboratory medium. Paired S. enterica serovar Typhi isolates recovered from the blood and bone marrow of individual patients exhibited similar PFGE patterns. Typing of S. enterica serovar Typhi isolates from patients with relapses of typhoid indicated that the majority of relapses were caused by the same S. enterica serovar Typhi strain that was isolated during the initial infection. However, some individuals were infected with distinct and presumably newly acquired S. enterica serovar Typhi isolates.     

Multicenter evaluation of epidemiological typing of methicillin-resistant Staphylococcus aureus strains by repetitive-element PCR analysis. The European Study Group on Epidemiological Markers of the ESCMID

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.     

Multilocus sequence typing versus pulsed-field gel electrophoresis for characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains are emerging pathogens. Molecular typing of ESBL-producing E. coli is useful for surveillance purposes, to monitor outbreaks and track nosocomial spread. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" for bacterial molecular typing, multilocus sequence typing (MLST) may offer advantages. Forty ESBL-producing E. coli isolates were selected at random from a cohort of intensive care unit patients who had active surveillance perirectal cultures done. PFGE identified 19 unique PFGE types (PT) among the 40 isolates; MLST identified 22 unique sequence types. MLST had greater discriminatory ability than PFGE for ESBL-producing E. coli. Simpson's indices of diversity for PFGE and MLST were 0.895 and 0.956, respectively. There were five clonal complexes (CCs) (isolates with differences of no more than two loci) that each contained multiple PT, but each PT was found in only one CC, indicating genetic consistency within a CC. MLST has clear utility in studies of ESBL-producing E. coli, based on a greater discriminatory ability and reproducibility than PFGE and the ability to a priori define genetically related bacterial strains.     

Diversification of Clonal Complex 5 Methicillin-Resistant Staphylococcus aureus Strains (Rhine-Hesse Clone) within Germany

Since 1995, a methicillin-resistant Staphylococcus aureus (MRSA) clone has spread in southern Germany. The strain was assigned to the Rhine-Hesse pulsed-field gel electrophoresis (PFGE) type by the staphylococcal reference center and was highly similar to epidemic clones known to belong to clonal complex 5 (CC5; USA100) based on multilocus sequence typing (MLST). Here we analyzed a defined collection of strains assigned to the Rhine-Hesse/USA100 PFGE type. Using sequence-based typing methods (MLST, spa), the isolates were divided into two distinct clusters, ST5 and its single-locus variant ST225. These two lineages are not distinguishable by PFGE or phage typing. Most of the ST5 isolates were derived from patients and volunteers from the Tübingen area in southwest Germany, whereas the ST225 isolates were mostly from other locations in Germany. The locally restricted ST5 isolates were shown to contain different SSCmec islands and exhibited different antibiotic resistance profiles. In contrast, the ST225 isolates form a highly homogenous group and are emerging all over Germany. The two lineages are clearly distinguishable by their phage content and spa type: ST5 strains from Tübingen are characterized by a Sa7int phage that carries the virulence gene sak, which codes for staphylokinase, and ST225 isolates are characterized by a Sa1int phage. In conclusion, based on sequence typing and phage content, CC5 strains can be subdivided into two distinct lineages with different epidemicities.     

Use of semi-quantitative and quantitative culture methods and typing for studying the epidemiology of central venous catheter-related infections in neonates on parenteral nutrition

To study the epidemiology - especially the impact of contaminated stopcocks - on central venous catheter (CVC) infection and catheter-related sepsis (CRS), semi-quantitative (SQ) and quantitative (Q) culture methods and typing of coagulase-negative staphylococci (CNS) were employed in 49 neonates with clinical signs of sepsis while receiving parenteral nutrition in the paediatric intensive care unit. The patients were divided into two groups according to stopcock contamination: group A consisted of 18 patients (36%) with contaminated stopcocks and group B consisted of 31 patients (64%) with sterile stopcocks. Five specimens were obtained from each patient, in addition to that from the stopcock: a swab taken from the skin surrounding the catheter puncture site; the CVC tip; the intradermal segment (IDC); and samples of parenteral fluid and blood. A total of 294 specimens (392 sites) was cultured and micro-organisms were identified. All CNS isolated were typed by biotyping, antibiogram, plasmid analysis and pulsed-field gel electrophoresis (PFGE), and the discriminatory power of the typing methods was compared. The CVC tips were infected in 25 patients (51%); 15 (83%) in group A and 10 (32%) in group B. Sepsis was detected in 24 neonates (49%), 13 in group A and 11 in group B. This was catheter-related in 15 patients (63%), 12 in group A and 3 in group B. CNS were recovered from 13 (52%) of 25 infected CVCs, nine in group A and four in group B. Sixty-five CNS isolates were recovered from these patients and belonged to 14 biotypes, 22 antibiograms, 22 plasmid profiles and 26 PFGE types. Typing showed that in six of nine patients in group A, CNS of the same type were recovered from the catheter tip and the stopcock, in one patient the catheter tip and skin isolates were the same and in two others the catheter tip isolates were different from stopcock and skin isolates. In all four patients in group B, different CNS types were recovered from CVC tips and skin. Bacteraemia was caused by CNS in 14 patients (58%), six in group A and eight in group B. Typing confirmed that nine cases (six in group A and three in group B) were catheter-related but five were not. SQ and Q culture methods and typing, especially by PFGE, allowed the study to determine that bacteria from contaminated stopcocks were frequently the source of CVC infection and CRS.     

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis

Objective   To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization.
Methods   In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme Spe I. For pyocin typing, the procedure described by Fyfe was applied.
Results   After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig.
Conclusions   Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.