烧伤病房铜绿假单胞菌的耐药检测及脉冲场凝胶电泳分析

目的了解烧伤病房铜绿假单胞菌的耐药性,用脉冲场凝胶电泳对铜绿假单胞菌进行分子流行病学的调查。方法采用琼脂纸片扩散法对9种抗菌药物作敏感试验,其中20株进行脉冲场凝胶分析。结果20株PAE对9种抗菌药物显示很高的耐药率,多重耐药率为30%;20株菌经脉冲场凝胶分析分别有3组菌株具有同源性。结论铜绿假单胞菌对常用抗菌药物耐药率高,多重耐药情况严重,脉冲场凝胶电泳是研究铜绿假单胞菌分子流行病学较好的基因分型方法。     

产超广谱β-内酰胺酶铜绿假单胞菌脉冲场电泳研究

目的：调查产超广谱β-内酰胺酶铜绿假单胞菌在医院烧伤病房中的流行情况及脉冲场电泳特征。方法：采用脉冲肠凝胶电泳技术对铜绿假单胞菌进行测定，并用双纸片扩散法筛选产超广谱β-内酰胺酶铜绿假单胞菌。结果：30株铜绿假单胞菌共筛选出7株（占23．3％）产超广谱β-内酰胺酶菌株；通过脉冲场指纹图谱并显示7株产超广谱β-内酰胺酶菌株都是同一基因型；其中3株来自烧伤科物体表面；4株来自烧伤病人创面；其对头孢他啶、头孢哌酮、头孢曲松、头孢噻肟、头孢吡肟、哌拉西林、亚胺培南、氨曲南、庆大霉素、妥布霉素、阿米卡星、环丙沙星和氧氟沙星等13种抗菌药物呈现全耐药现象。结论：在医院感染产超广谱β-内酰胺酶铜绿假单胞菌的分子流行病学研究中，脉冲场凝胶电泳是一种较好的研究方法。     

烧伤病房铜绿假单胞菌脉冲场电泳分型及耐药性研究

目的：了解医院烧伤病房铜绿假单胞菌的基因型及耐药性。方法：采用脉冲场凝胶电泳技术对铜绿假单胞菌进行基因分型，结合药物敏感试验进行分析。结果：30株铜绿假单胞菌的脉冲场谱分10型（A—K型），对13种抗生素的耐药性由高到低为庆大霉素（67％）、阿米卡星（67％）、环丙沙星（64％）、氧氟沙星（64％）、头孢噻肟（50％）、头孢曲松（44％）、妥布霉素（40％）、氨曲南（30％）、头孢哌酮（27％）、头孢吡肟（24％）、哌拉西林（24％）、头孢他啶（23％）、亚胺培南（23％）。筛选出了7株（占23．3％）产超广谱β-内酰胺酶菌株，它们对13种抗生素出现全耐药现象，指纹图谱显示均是同一基因型（D型）。结论：在医院感染铜绿假单胞菌的分子流行病学研究中，脉冲场凝胶电泳是一种较好的基因分型方法。     

2010年食物中毒及散发腹泻患者沙门菌PFGE分子分型研究

目的 研究2010年沙门菌食物中毒及散发腹泻患者沙门菌分离株的分子分型.方法 将从中毒事件患者和剩余食物及散发腹泻患者分离到的18株沙门菌进一步鉴定培养,用限制性内切酶Xba l消化酶切后进行脉冲场凝胶电泳,运用Bionumerics5.1聚类分析.结果 指纹图谱聚类显示18株沙门菌分为多个分子型,同起食物中毒分离到的沙门菌有相同的指纹图谱;来自两个散发腹泻患者的分离株图谱也一致.结论 脉冲场凝胶电泳可有效应用于食物中毒溯源分析及沙门菌分子流行病学研究.     

两起伤寒沙门菌感染暴发流行病原的相似度研究

目的 应用脉冲场凝胶电泳(PFGE)技术对两起由伤寒沙门菌引起的聚集性暴发流行中所分离到的伤寒沙门菌进行分离鉴定和基因分型,对其病原进行同源性分析和分子流行病学探讨,为科学防治提供实验室检测依据. 方法 用传统方法对11株伤寒沙门菌菌株进行生化、血清学复核鉴定.对确认的伤寒沙门菌菌株,采用脉冲场凝胶电泳技术分析电泳酶切指纹图谱. 结果 11株伤寒沙门菌经PFGE,根据其条带的差异可分为3个PFGE型别. 结论 两起伤寒沙门菌感染为不同亚型所致.     

应用脉冲场凝胶电泳对伤寒沙门菌分型研究

目的:运用脉冲场凝胶电泳(PFGE)技术对湖北2008年分离的沙门菌菌株进行分析,探讨湖北沙门菌的分布、流行及变异情况。方法:对分离的沙门菌利用全自动生化分析系统、血清分型、及脉冲场凝胶电泳(PFGE)分子生物学技术进行研究。结果:18份测试株用XbaI酶切后产生3个带型。结论:通过脉冲场凝胶电泳(PFGE)全基因DNA指纹图谱分析,可追溯其同源性。通过18株伤寒菌株的PFGE分型图谱分析,其中相似系数100%15份,占83.33.%,可确认为相同病原菌感染。由此说明PFGE是一种灵敏、可靠的研究沙门菌分子流行病学基因分型方法。     

山东省2014年矿泉水中微生物检测及铜绿假单胞菌分离株脉冲场凝胶电泳分型

目的初步掌握山东省矿泉水中微生物的污染状况,并建立铜绿假单胞菌PFGE指纹图谱,为卫生监督和流行病学调查提供依据。方法依据GB/T 8538—2008《饮用天然矿泉水检验方法》对矿泉水进行检验,利用PFGE分型技术对26株铜绿假单胞菌进行全基因组分析,用限制性内切酶SpeⅠ切割,PFGE技术建立PFGE指纹图谱,用Bio Numerics V 5.1软件进行聚类分析。结果所有水样中除铜绿假单胞菌外的3种微生物指标的合格率均为100.00%,铜绿假单胞菌的合格率为56.60%。26株菌的PFGE图谱的相似度为65%~100%,经聚类分析得到12种PFGE基因指纹图谱。其中K有6株菌相似度为100%,为三地市铜绿假单胞菌的优势型别。结论山东省部分地区矿泉水中铜绿假单胞菌的污染严重,有必要建立有效的卫生监督机制。     

福建省鼠伤寒沙门菌分子流行病学研究

目的了解福建省鼠伤寒沙门菌脉冲场凝胶电泳(PFGE)分子分型情况,探讨各型别的流行及分布特征。方法收集2009-2010年鼠伤寒沙门菌73株,分离自临床患者及从业体检人员,选择XbaⅠ和BlnⅠ进行酶切,H9812作为脉冲场凝胶分子量标准。采用脉冲场凝胶电泳技术分析电泳酶切指纹图谱,运用BioNumerics软件进行聚类分析。结果按照100%的相似度可将菌株酶切图谱分为49个PFGE型,其中P1型有15株,占20.55%(15/73),为优势型别,P17型5株,占6.85%;BlnⅠ二次酶切后P1和P17型分别只有5株和2株条带完全一致。P1型集中分布于7～9月,占到P1型菌株总数的86.67%(13/15);其他型别分布比较分散,无明显时间和地区的聚集性。结论福建省鼠伤寒沙门菌PFGE分子分型呈现多态性,该技术可用于菌株间分子流行病学研究,应加强监测的时效性和针对性以利于疾病的溯源和早期预警。     

烧伤病房铜绿假单胞菌耐药谱检测及分子流行病学研究

目的了解分离自烧伤病房铜绿假单胞菌耐药表型及分子流行病学特性。方法对分离自烧伤病房的32株铜绿假单胞菌采用K-B法测定抗菌药物的敏感性;利用碱裂解法提取质粒获得质粒DNA图谱;利用脉冲场电泳分析菌株的亲缘性。结果 32株铜绿假单胞菌中有17株对8种抗菌药物耐药率,占53.1%,8株(25.0%)铜绿假单胞菌对检测抗菌药物全部耐药;29株(90.6%)含有质粒,分属于4种质粒表型,主要有2种质粒表型的铜绿假单胞菌;脉冲场电泳聚类分析显示铜绿假单胞菌感染主要由3个克隆株引起。结论分离自烧伤病房的铜绿假单胞菌耐药严重,医院感染铜绿假单胞菌可导致克隆传播,甚至暴发流行。     

2007年四川省鼠伤寒沙门菌PFGE分型及溯源

目的 用脉冲场凝胶电泳(PFGE)分型技术.研究四川省2007年鼠伤寒沙门菌分子流行病学,查找沙门菌污染源,为预测疫情和制定防治措施提供依据.方法 选择Xba I酶对12株鼠伤寒沙门菌全基因组DNA进行酶切,用PFGE对菌株进行分子分型,Bionumerisc统计软件聚类分析.结果 12株鼠伤寒沙门菌用XbaI限制性酶切后,分成5个PFGE图谱类型,其中1个PFGE图谱类型有7株菌株,其指纹图谱的相似性达到100%,该型别占分析菌株的58.33%.其余4个PFGE图谱类型分别有2株菌和1株菌.含7株菌的PFGE图谱型中,源于成都市的菌株4株,攀枝花、自贡和内江市的菌株各1株.结论 PFGE分子分型与流行病学资料紧密结合可增强对鼠伤寒沙门菌食源性疾病的溯源和预警.     

阪崎肠杆菌脉冲场凝胶电泳分型的研究

目的对29株阪崎肠杆菌分离株和2株阪崎肠杆菌模式株进行脉冲场凝胶电泳(PFGE)分型研究。方法分别利用限制性内切酶XbaⅠ和SpeⅠ酶切阪崎肠杆菌染色体DNA进行PFGE分析,利用BioNumerics软件进行聚类分析。结果31株阪崎肠杆菌使用XbaⅠ和SpeⅠ酶切分别有30种PFGE带型。除ES004和ES005外,其他29株阪崎肠杆菌经XbaⅠ或SpeⅠ酶切后的PFGE条带之间均存在不同程度的差异。来源于同品牌同批次中不同包装的产品中的ES004和ES005分离株XbaⅠ和SpeⅠ酶切图谱完全一致(相似度为100%)。根据BioNumerics软件分析结果可知,除分离株ES004和ES005外,其他分离株的带型和样品来源之间无显著相关性。结论虽然XbaⅠ酶切的PFGE带型平均条带少于SpeⅠ,但对阪崎肠杆菌具有足够的分辨率。两种PFGE分型方法都可应用于阪崎肠杆菌的分子分型和溯源。     

Clinical surveillance of surgical imipenem-resistant Pseudomonas aeruginosa infection in a Japanese hospital

In order to elucidate any changes in imipenem-resistant Pseudomonas aeruginosa (IRPA) infections in Japan, we examined 511 P. aeruginosa stains isolated from our surgical ward between 1987 and 2001. These isolates were subjected to susceptibility testing against various antipseudomonal agents including imipenem, meropenem, ceftazidime, gentamicin and ciprofloxacin. They were serotyped with the slide agglutination test and genotyped using pulsed-field gel electrophoresis (PFGE). The annual incidences of IRPA infections were particularly high in the early 1990s. Epidemiological investigations revealed that these outbreaks were due to dissemination of hospital-acquired IRPA isolates. Intensive use of imipenem promoted the selection of highly resistant strains. Further study of resistance mechanisms revealed that none of the 110 IRPA strains were metallo-beta-lactamase (MBL) producers. Polymerase chain reaction (PCR) analysis using bla(IMP) specific primers confirmed that no IMP-1 type MBL gene-positive strains were detected from our ward. Susceptibilities of those IRPA strains against other antipseudomonal agents showed relatively low levels, suggesting that imipenem resistance was mainly due to impermeability of the OprD porin. In conclusion, hospital-acquired outbreaks of IRPA were recently reduced by guidelines for, and surveillance of, appropriate use of antimicrobial agents. When the rate of IRPA isolation increases, serotyping should be performed initially and PFGE is required to confirm outbreaks. A computer-assisted genotyping technique is available to perform epidemiological studies of IRPA isolates.     

Clonal identification of Aeromonas hydrophila strains using randomly amplified polymorphic DNA analysis

The suitability of arbitrary primer polymerase chain reaction (RAPD) as a typing technique was evaluated by comparing it with pulsed-field gel electrophoresis (PFGE) to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections. Five isolates from patients and 10 isolates from the water supply were compared to 10 epidemiologically unrelated strains isolated from patients and rivers. Two methods were used to prepare DNA and two primers (AP3 and AP5) were selected. The discriminatory power was better with the extractive DNA preparation than the boiling method. The discrimination of closely related from less related strains by PCR using AP3 was consistent with that by PFGE: water supply of Cholet hospital contaminated with Aeromonas species was not the source of the cluster of hospital infections and only two patients were infected with clonally-related strains. RAPD using primer AP3 was simpler, cheaper, and quicker to perform than pulsed-field gel electrophoresis and is well suited for the epidemiological study of A. hydrophila isolates.     

Detection of type III secretion system virulence and mutations in gyrA and parC genes among quinolone-resistant strains of Pseudomonas aeruginosa isolated from imported shrimp

Twenty Pseudomonas aeruginosa isolates were recovered from imported frozen raw shrimp sold in the United States. Isolates were tested for antimicrobial susceptibility to quinolones and analyzed for mutations in quinolone resistance-determining regions, presence of type III secretion system genes, and genetic relatedness using pulsed-field gel electrophoresis. All isolates were resistant to nalidixic acid. Polymerase chain reaction assays detected exoS, exoT, exoU, and exoY among isolates. Eight unique pulsed-field gel electrophoresis clusters were generated. Mutations were found in gyrA at codon 83 (Ile to Thr) and in parC at codon 87 (Leu to Ser). Together, these findings reveal that imported shrimp may harbor virulent and quinolone-resistant strains of P. aeruginosa.     

Pseudomonas aeruginosa and the oropharyngeal ecosystem of tube-fed patients

We evaluated whether elderly patients fed with nasogastric tubes (NGT) are predisposed to Pseudomonas aeruginosa colonization in the oropharynx. Fifty-three patients on NGT feeding and 50 orally fed controls with similar clinical characteristics were studied. The tongue dorsum was swabbed and cultured. P. aeruginosa was isolated in 18 (34%) of the NGT-fed group but in no controls (p<0.001). Other gram-negative bacteria were cultured from 34 (64%) of NGT-fed patients as compared with 4 (8%) of controls (p<0.001). Antibiotic susceptibility of the oropharyngeal P. aeruginosa isolates was compared with that of isolates from sputum cultures obtained from our hospital's bacteriologic laboratory. The oropharyngeal isolates showed a higher rate of resistance; differences were significant for amikacin (p<0.03). Scanning electron microscope studies showed a biofilm containing P. aeruginosa organisms. The pulsed-field gel electrophoresis profile of these organisms was similar to that of P. aeruginosa isolates from the oropharynx. NGT-fed patients may serve as vectors of resistant P. aeruginosa strains.     

重复片段PCR检测葡萄球菌DNA指纹在医院感染中的分析

目的 应用重复片段PCR，对凝固酶阴性葡萄球菌(CNS)进行基因分型，用于医院感染中分子流行病学研究。方法 选择REPl REP2和ERICl ERIC2两对引物，对临床分离的68株(CNS)菌株进行重复片段PCR，根据扩增产物的电泳模式编制菌株问的相似矩阵，并利用RAPD、PHYLIP及TREEVIEw等软件，绘制各株间的遗传聚类图。结果 两对引物的扩增带型均比较丰富，对于实验CNS菌株具有较好的分辨率，根据聚类结果，可以确定某些菌株之间的同源性，为感染源的确认等分子流行病学研究提供依据。结论 重复片段PCR分辨率高，根据聚类结果确定菌株的同源性，为医院感染确认感染源提供了分子流行病学的依据。     

应用PFGE及SPA-typing基因分型技术对金葡菌食物中毒溯源分析

目的建立金黄色葡萄球菌（金葡菌）的脉冲场凝胶电泳（PFGE）和SPA基因序列分析技术（SPA-typing）,应用于金葡菌引起的食物中毒准确溯源。方法按照GB/T4789-2003对样品进行菌株分离和生化鉴定。对同一起食物中毒分离的金葡菌株,分别用PFGE和SPA-typing技术进行分析。结果在食物中毒调查中共7份样品分离出金葡菌。该批菌株经PFGE分型呈现为一致的带型,相似性系数为100%。SPA-typing分型均为t 5593型。结论 PFGE和SPA-typing均显示患者、剩余食品和加工环境来源菌株均为同一克隆群。这是一起由于加工环境污染金葡菌引起的食物中毒。PFGE和SPA-typing分型技术均显示了良好的分型效果。     

Genomic fingerprinting of shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment-length polymorphism (FAFLP)

For epidemiological studies of shigatoxin-producing Escherichia coli (STEC) infections, rapid, reproducible and highly discriminative methods are required. In this study, we examined the performance of the fluorescent amplified-fragment-length polymorphism (FAFLP) technique for epidemiological fingerprinting of STEC isolates and compared it to the acknowledged fingerprinting method pulsed-field gel electrophoresis (PFGE). A total of 88 STEC isolates, including 82 of serotype O157:H7 or O157:H-, were subjected to fingerprinting by both PFGE and FAFLP. The isolates included sporadic and epidemiologically related strains of both animal and human origin from widespread geographical locations. The FAFLP fingerprint patterns confirmed the clonal nature of STEC O157 strains. Among the 82 O157:H7/H- isolates belonging to 49 distinct groups of epidemiological unrelated isolates, 24 FAFLP profiles and 51 PFGE patterns were obtained. Thus, PFGE had a higher discriminatory power than FAFLP and overall correlated better to available epidemiological data. Consequently, the PFGE technique remains the method of choice in epidemiological investigations of STEC infections.     

Epidemiological analysis of carbapenem-sensitive and -resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa with decreased levels of meropenem susceptibility were identified in the Royal Infirmary Edinburgh in 2002. Within the affected group of patients, none had meropenem-resistant P. aeruginosa when they arrived in the intensive care unit (ICU). Seven isolates from the ICU were collected five months after the decreased susceptibility to meropenem was identified. In order to investigate if resistance was a problem in P. aeruginosa throughout Edinburgh, both in hospital- and community-acquired isolates, a prospective study was performed. The susceptibilities of 104 P. aeruginosa to imipenem, meropenem, ceftazidime, piperacillin/tazobactam and ciprofloxacin were investigated. Meropenem had the highest activity against these isolates and the lowest MIC(90) (2 mg/L), followed by imipenem (4 mg/L), ciprofloxacin (8 mg/L), piperacillin/tazobactam (16 mg/L) and ceftazidime (32 mg/L). These isolates were also analysed genotypically by pulsed-field gel electrophoresis. Five of the seven ICU isolates were identified, one isolate was 98% similar and the other was 85% similar to the ICU isolates. One isolate from the prospective study had approximately 90% genotype similarity to the six ICU isolates with >/=98% similarity. There was no clonality within the strains from the prospective study and clusters with >90% similarity comprised at five or less isolates. Isolates with the same resistance patterns did not necessarily have the same genotypic profile. Strains isolated from different patients on the same day were also not necessarily related. The conclusions of this study were that while the seven ICU isolates were clonal or highly related, they were not widespread throughout Edinburgh and the P. aeruginosa within Edinburgh were highly varied.